Methods and Compositions for Delivery of Catecholic Butanes for Treatment of Tumors

ABSTRACT

The present invention provides kits, methods and compositions for the treatment of diseases such as cancers. The compositions herein contain a substantially pure preparation of at least one catecholic butane, including, for example, NDGA compounds in a pharmaceutically acceptable carrier or excipient. The catecholic butane such as NDGA or its derivatives are administered to one or more subjects in need of treatment by a route other than direct injection into the affected tissues or topical application on affected tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/284,111 filed Dec. 4, 2007, now U.S. Pat. No. 7,728,036, which is acontinuation of International Patent Application No. PCT/US2004/016235,filed May 20, 2004, and published in the English language asInternational Publication No. WO 2005/007080 on Jan. 27, 2005, whichclaims priority to U.S. provisional application No. 60/472,008, filedMay 20, 2003; U.S. provisional application No. 60/472,144, filed May 20,2003; U.S. provisional application No. 60/472,188, filed May 20, 2003;U.S. provisional application No. 60/472,282, filed May 20, 2003; andU.S. provisional application No. 60/472,299, filed May 20, 2003; thecontents of all of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

This invention relates to kits, methods and compositions containingcatecholic butanes for the delivery of such to subjects for thetreatment of malignant, premalignant and benign tumors. This inventionalso relates to methods of making the foregoing compositions. In suchmethods of treatment, one or more catecholic butanes are administered tosubjects via routes of delivery other than direct injection into theaffected tissue, and other than topical application onto the affectedtissue. This invention further relates to compositions comprising one ormore catecholic butanes that are formulated appropriately for such modesof delivery and treatment.

Catecholic butanes, including nordihydroguaiaretic acid (“NDGA”) and itsderivatives, have been used for the inhibition of tumor growth incertain experimental animals. For example, Jordan et al. in U.S. Pat.No. 5,008,294 described the use of a single dose of NDGA on a mammarycarcinoma MX-1 xenograft in athymic nude NCr mice. In one experiment,NDGA was injected into the tumor one day following subcutaneousimplantation of a 14 mg fragment of the human mammary carcinoma in theaxillary region of the mice. Jordan et al. further described topicalapplication of NDGA after day 23 of implantation of human breastadenocarcinomas in athymic mice. Some evidence of inhibition of tumorgrowth was observed in those experiments, but it is unclear whether theantitumor effect was durable.

Huang et al. in U.S. Pat. No. 6,417,234 and U.S. Pat. No. 6,214,874described intratumor injection of a NDGA derivative, designatedtetra-O-methyl NDGA or M₄N, and another NDGA derivative, designated G₄N,separately or together into mice implanted with HPV-16 transformedimmortal mouse epithelial cells (C3). Huang et al. also found someevidence of suppression of tumor growth by these NDGA derivatives. It isunknown whether compounds such as these NDGA derivatives can be safelyadministered to other animals such as humans.

Certain of the catecholic butanes, such as M₄N, which is a NDGAderivative, are hydrophobic compounds found to be soluble in dimethylsulfoxide (“DMSO”). When the composition of M₄N in DMSO was injectedinto the tumor, the composition appeared to penetrate most but not allof the tumor tissues. A possible explanation may be that the hydrophobicnature of the compound limits its penetration. It would be desirable ifa formulation can be found for safe systemic administration of thesehydrophobic compounds so as to improve their efficacy, expand theirutility and yet maintain their biological activities, such as anti-tumoractivities. It would further be desirable if the catecholic butanes,including the NDGA Compounds, can be safely administered by routes ofadministration other than by direct injection into the affected tissuesor by topical application.

Moreover, intratumor injection or direct injection of drugs intoaffected tissues may not be an ideal treatment regimen. Patientssometimes experience injection site discomfort. In addition, many tumorsare not amenable to intratumor injection of a therapeutic, and many maynot respond to topical application of a therapeutic. It would bedesirable if a different route of administration of these catecholicbutanes can be found that would be safe and appropriate for the diseaseor condition and yet maintain the biological activities of suchcompounds.

Additionally, it is not known whether the catecholic butanes, includingNDGA and NDGA derivatives (collectively, the “NDGA Compounds”), orformulations containing them can differentially inhibit the growth orprogression of tumor growth in humans without adversely affecting normaltissues. It would be desirable if the catecholic butanes, including theNDGA Compounds can be formulated and administered in such a way as tospare the normal tissues of any adverse effects.

Further, a majority of human malignant tumors are both local andsystemic in nature in that the primary malignant tumors are producedlocally whereas the secondary tumors, seeded from the primary, arespread systemically to other tissues, or arise de novo from tissuessimilar to the source of the primary. The most appropriate therapeuticoptions, therefore, include those that deliver effective medication tothe primary source of malignancy as well as to the secondary sources. Itwould be desirable if an effective therapeutic can be formulated thatcan access both the primary and secondary sources of malignancies.

It would also be desirable if the NDGA derivatives can be formulated ina manner that would facilitate delivery to targeted tissues andmaintenance of a certain range of dose level in the targeted tissues.

BRIEF SUMMARY OF THE INVENTION

It is, thus, one of the objects of the present invention to providemethods and compositions for the prevention or treatment of tumors suchas to address the problems in the prior art methods and compositions,for example, those described in the Background.

It is another one of the objects of the present invention to providemethods and compositions as above, such as, for example, to inhibit thegrowth, development or progression of tumors.

It is another one of the objects of the present invention to provide oneor more methods of administering the catecholic butanes, including theNDGA Compounds, that are effective in the prevention or treatment oftumors as above, where the targeted tissues or the affected tissues tobe treated are not easily accessible to direct injection or amenable totopical application of such compounds.

It is a further one of the objects of the present invention to provideone or more formulations containing the catecholic butanes, includingthe NDGA Compounds, that can facilitate and/or optimize distribution ofthe catecholic butanes, including the NDGA Compounds, to the targetedtissues.

It is another one of the objects of the present invention to providecompositions containing one or more catecholic butanes, including theNDGA Compounds, in formulations appropriate for treatment of thetargeted tissues.

In accordance to one of the objects of the present invention, there isprovided a pharmaceutical composition for treatment of a disease in asubject, such as an animal, for example, a human, where the compositioncontains at least one catecholic butane and a pharmaceuticallyacceptable carrier or excipient, and where the composition is formulatedfor administration by a route other than by direct injection into ortopical application onto an affected tissue.

In accordance to another one of the objects, there is provided acomposition as above, where the disease, disorder or condition is otherthan an inflammatory disease, for example, other than an inflammatorydisease that is associated with microglial cell activation orstimulation.

In accordance to another one of the objects, there is provided acomposition as above, where the disease is a proliferative disease. Sucha proliferative disease may be a malignant tumor, a premalignantcondition, or a benign tumor.

In accordance to a further one of the objects, there is provided acomposition as above, where the disease results from or is associatedwith a virus infection, such as, for example, HIV infection, HPVinfection, or HSV infection.

In accordance to still another one of the objects, there is provided acomposition as above, where the composition is formulated for intranasaladministration, oral administration, including through slow release orrapid release capsules, for inhalation, for subcutaneous administration,for transdermal administration, for intra-arterial administration, withor without occlusion, for intracranial administration, intraventricularadministration, intravenous administration, buccal administration,intraperitoneal administration, intraocular administration, centralvenous administration, intramuscular administration or for implantation.

In accordance to another one of the objects, there is provided acomposition as above, where the pharmaceutically acceptable carrier orexcipient contains dimethyl sulfoxide (DMSO), phosphate buffered saline(PBS), saline, an oil such as, for example, castor oil or corn oil,Cremaphor EL, and ethanol or a mixture containing one or more of such.

In accordance to another one of the objects, there is provided acomposition as above, where the pharmaceutically acceptable carrier orexcipient contains a lipid based formulation, a liposomal formulation, ananoparticle formulation, a micellar formulation, a water solubleformulation, a Cremaphor EL/ethanol/saline formulation or any of theforegoing in a biodegradable polymer.

In accordance to yet another one of the objects, there is provided acomposition as above, where the catecholic butane has the structuralformula I as follows:

where R₁ and R₂ are independently —H, a lower alkyl, a lower acyl, analkylene or an unsubstituted or substituted amino acid residue or saltthereof; R₃, R₄, R₅, R₆, R₁₀, R₁₁, R₁₂ and R₁₃ are independently —H or alower alkyl; and R₇, R₈ and R₉ are independently —H, —OH, a loweralkoxy, a lower acyloxy, or any two adjacent groups together may be analkyene dioxy, or an unsubstituted or substituted amino acid residue orsalt thereof.

In accordance to still another one of the objects, there is provided acatecholic butane as above, where R₁ and R₂ are independently —H, alower alkyl, a lower acyl, or an unsubstituted or substituted amino acidresidue or salt thereof; R₃, R₄, are independently a lower alkyl; R₅,R₆, R₁₀, R₁₁, R₁₂ and R₁₃ are independently —H; and R₇, R₈ and R₉ areindependently —H, —OH, a lower alkoxy, a lower acyloxy, or unsubstitutedor substituted amino acid residue or salt thereof.

In accordance to yet another one of the objects, there is provided acatecholic butane as above, where R₁ and R₂ are independently —H, alower alkyl, a lower acyl, or an unsubstituted or substituted amino acidresidue or salt thereof; R₃, R₄, are independently a lower alkyl; R₅,R₆, R₇, R₁₀, R₁₁, R₁₂ and R₁₃ are independently —H; and R₈ and R₉ areindependently —OH, a lower alkoxy, lower acyloxy, or an unsubstituted orsubstituted amino acid residue or salt thereof.

In accordance to still another one of the objects, there is provided thecatecholic butane as above, where R₁ and R₂ are independently —CH₃ or—(C═O)CH₂N(CH₃)₂ or a salt thereof.

In accordance to still another one of the objects, there is provided thecatecholic butane as above, where R₈ and R₉ are independently —OCH₃ or—O(C═O)CH₂N(CH₃)₂ or a salt thereof.

In accordance to still another one of the objects, there is provided thecatecholic butane as above, where R₁ and R₂ are independently —CH₃,—(C═O)CH₂N(CH₃)₂ or —(C═O)CH₂N⁺H(CH₃)₂.Cl⁻ and R₈ and R₉ areindependently —OCH₃, —O(C═O)CH₂N(CH₃)₂ or —O(C═O)CH₂N⁺H(CH₃)₂.Cl⁻.

In accordance to still another one of the objects, there is provided thecatecholic butane as above, where R₁ and R₂ are independently —H or —CH₃and R₈ and R₉ are independently —OH or —OCH₃, provided that thecatecholic butane is not NDGA.

In accordance to still another one of the objects, there is provided thecatecholic butane as above, where R₁ and R₂ are independently —CH₃ andR₈ and R₉ are independently —OCH₃.

In accordance to still another one of the objects, there is provided thecatecholic butane as above, where the catecholic butane is NDGA.

In accordance to still another one of the objects, there is provided thecatecholic butane as above, where the catecholic butane is other thanNDGA.

In accordance to yet another one of the objects, there is provided amethod of making a pharmaceutical composition containing a catecholicbutane, where the method includes the steps of (a) providing acatecholic butane as above; (b) providing a pharmaceutically acceptablecarrier or excipient as above, and (c) combining the catecholic butanewith the pharmaceutically acceptable carrier or excipient.

In accordance to a further one of the objects of the present invention,there is provided a method of treating a disease in a subject, where themethod of treatment includes providing a pharmaceutical composition asabove and administering the composition to the subject by a route otherthan by direct injection into the tumor or topical application onto thetumor.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the disease is other than an inflammatorydisease, for example, other than an inflammatory disease that isassociated with microglial cell activation or stimulation.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the disease is a proliferative disease suchas a malignant tumor, a premalignant condition, or a benign tumor.

In accordance to a further one of the objects, there is provided amethod of treatment as above, where the disease results from or isassociated with a virus infection, such as, for example, HIV infection,HPV infection, or HSV infection.

In accordance to still another one of the objects, there is provided amethod of treatment as above, where the composition is formulated forintranasal administration, oral administration, including through slowrelease or rapid release capsules, for inhalation, for subcutaneousadministration, for transdermal administration, for intra-arterialadministration, with or without occlusion, for intracranialadministration, intraventricular administration, intravenousadministration, buccal administration, intraperitoneal administration,intraocular administration, central venous administration, intramuscularadministration or for implantation.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the pharmaceutically acceptable carrier orexcipient contains dimethyl sulfoxide (DMSO), phosphate buffered saline(PBS), saline, an oil such as, for example, castor oil or corn oil,Cremaphor EL, ethanol and any combination of such.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the pharmaceutically acceptable carrier orexcipient contains a lipid based formulation, a liposomal formulation, ananoparticle formulation, a micellar formulation, a water solubleformulation, a Cremaphor EL/ethanol/saline formulation or any of theforegoing in a biodegradable polymer.

In accordance to yet another one of the objects, there is provided amethod of treatment as above, where the catecholic butane has a formulagiven above.

In accordance to yet another one of the objects, there is provided amethod of treatment as above, where the catecholic butane istetra-O-methyl NDGA.

In accordance to still another one of the objects, there is provided amethod of treatment as above, where the catecholic butane istetra-dimethylglycinyl NDGA.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is tri-O-methyl NDGA.

In accordance to still another one of the objects, there is provided amethod of treatment as above, where the catecholic butane is NDGA.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is other than NDGA.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the method includes administering at leasttwo catecholic butanes.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the two catecholic butanes are administeredsubstantially contemporaneously.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the two catecholic butanes are administeredat different times.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the two catecholic butanes are selectedfrom the group consisting of tetra-O-methyl NDGA, tri-O-methyl NDGA andtetra-dimethylglycinyl NDGA.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the nanoparticle formulation contains atleast one selected from the group consisting ofpoly(DL-lactide-co-glycolide), poly vinyl alcohol, d-α-tocopherylpolyethylene glycol 1000 succinate, andpoly(lactide-co-glycolide)-monomethoxy-poly(polyethylene glycol).

In accordance to another one of the objects, there is provided a methodof treatment as above, where the liposomal formulation comprises atleast one selected from the group consisting ofphosphatidylcholine/cholesterol/PEG-DPPE,distearoylphosphatidylcholine/cholesterol/PEG-DPPE, and1-2-dioleoyl-sn-glycero-3-phosphocholine/1-2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)sodium salt/cholesterol/triolein/tricaprylin.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the disease is cancer and the cancer is asolid tumor, a lymphoma or leukemia.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the cancer is selected from the groupconsisting of malignant, pre-malignant or benign brain tumor, nasalpharyngeal tumor, head and neck tumor, liver tumor, kidney tumor,prostate tumor, breast tumor, a bladder tumor, pancreatic tumor, stomachtumor, colon tumor, ovarian tumor, cervical tumor, and skin tumor andmetastases thereto.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the method comprises administering thecomposition more than once.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the pharmaceutically acceptable carrier orexcipient is an aqueous preparation.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the pharmaceutically acceptable carrier orexcipient comprises a hydrophobic preparation.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the hydrophobic preparation comprises alipid based vehicle.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the pharmaceutically acceptable carrier orexcipient comprises at least one selected from the group consisting ofcastor oil, peanut oil, dimethyl sulfoxide (DMSO), and other dietaryfats or oils.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the composition is formulated in the formof one selected from the group consisting of a tablet, a powder, a gelcapsule, a liquid, and an oral rinse.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the pharmaceutically acceptable carrier orexcipient comprises a polymer formulation.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the polymer formulation is a biodegradablepolymer formulation.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the pharmaceutically acceptable carrier orexcipient allows for high local drug concentration and sustained releaseover a period of time.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the polymer formulation comprises at leastone selected from the group consisting of 1,3-bis(p-carboxyphenoxy)propane, sebacic acid, poly(ethylene-co-vinyl acetate), andpoly(lactide-co-glycolide).

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is dissolved insaline, DMSO or ethanol prior to administration.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the composition is at least one selectedfrom the group consisting of: a powder, an aerosol, an aqueousformulation, a liposomal formulation, a nanoparticle formulation, and ahydrophobic formulation.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the composition is administered daily for adefined period of time.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the composition is administeredintermittently.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is infused into thesubject.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is a water solublecompound.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is a hydrophobiccompound.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is formulated as aliquid, an aerosol, an oral rinse, a suspension, a tablet, a powder, ora gel capsule.

In accordance to yet one of the objects, there is provided a method oftreatment of a viral infection in a subject comprising administering thecomposition of claim 1 to the subject, wherein the viral infectionresults from or is associated with HIV, HPV, or HSV.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is administered in arange of greater than about 10 mg/kg and less than about 375 mg/kg perdose into humans.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the range is greater than about 10 mg/kgand less than about 250 mg/kg per dose.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the range is greater than about 10 mg/kgand less than about 200 mg/kg per dose.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the range is greater than about 10 mg/kgand less than about 150 mg/kg per dose.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the range is greater than about 10 mg/kgand less than about 100 mg/kg per dose.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the range is greater than about 10 mg/kgand less than about 75 mg/kg per dose.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the range is greater than about 10 mg/kgand less than about 50 mg/kg per dose.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the composition is administeredsystemically, such as intravenously, for example.

In accordance to another one of the objects, there is provided a methodof treatment as above, where the catecholic butane is tri-O-NDGA ortetra-O-methyl NDGA.

In accordance to still one of the objects, there is provided a kit fortreatment of a disease comprising the pharmaceutical composition aboveand instructions for administration of the composition.

In accordance to a further one of the objects, there is provided amethod of treating a tumor in a subject, where the tumor is a malignant,premalignant or benign tumor, and where the tumor arises from or isassociated with a tissue or organ selected from the group consisting of:breast, liver, stomach, pancreas, colorectal, colon and prostate,comprising the steps of: (a) providing a composition containingtetra-O-methyl NDGA (M₄N) and a pharmaceutically acceptable carrier orexcipient; and (b) administering the composition to the subject; wherethe composition is administered other than by direct injection into ortopical application onto the tumor.

In accordance to a further one of the objects, there is provided amethod of treating a tumor as above, where the method includesadministering the composition orally, where the oral composition may bea slow release formulation or a rapid release formulation.

In accordance to a further one of the objects, there is provided amethod of treating a tumor as above, where the pharmaceuticallyacceptable carrier or excipient is an oil, such as, for example, castoroil or corn oil.

In accordance to a further one of the objects, there is provided amethod of treating a tumor as above, where the composition is present inan edible mix.

In accordance to a further one of the objects, there is provided amethod of treating a tumor as above, where the catecholic butanecomposition is administered daily for a period of time, such as, forexample, daily for 5 or more days to a week, or daily for 5 or more daysto 2 weeks, or daily for 5 or more days to 3 weeks.

In accordance to a further one of the objects, there is provided amethod of treating a tumor as above, where the amount of tetra-O-methylNDGA administered is at least 30 mg per dose, or optionally, at least 90mg per dose.

In accordance to a still further one of the objects, there is provided amethod of treating a tumor as above, where tetra-O-methyl NDGA ispresent in the composition at a concentration of 20 mg/mL.

In accordance to a further one of the objects, there is provided amethod of treating a tumor as above, where the pharmaceuticallyacceptable carrier or excipient comprises Cremaphor EL, ethanol andsaline, where Cremaphor EL may be present at a concentration of about6%, ethanol may be present at a concentration of about 6%, and salinemay be present at a concentration of about 88%, for example.

In accordance to another one of the objects, there is provided a methodof treating a tumor as above, where the composition administered to thesubject comprises at least 2 mg of tetra-O-methyl NDGA per dose.

In accordance to a further one of the objects, there is provided amethod of treating a tumor as above, where the composition isadministered intravenously or intraperitoneally.

In accordance to a still another one of the objects, there is provided amethod of treating a tumor as above, where the composition isadministered more frequently than once every 6 days for a period of timeor optionally, more frequently than once every 2 days for a period oftime.

Further objects, features and advantages of the present invention willbe apparent to one of ordinary skill in the art upon reading the presentdescription. Such other objects, features, and advantages are alsodeemed embodied by the present invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

In the drawings:

FIG. 1 shows systemic distribution of M₄N to various organs at 3 hoursfollowing intravenous and intraperitoneal injection. Mice were injectedwith 100 μCi of ³H— M₄N and 60 mM of unlabeled M₄N. Organs and bloodwere harvested and weighed at 3 hours post-injection and the M₄N wasextracted. The tritium content of the organ extracts were measured, andthe quantity of M₄N in each organ was calculated based on the specificactivity of the inoculum. FIG. 1A represents the quantity of M₄N, inmicrograms per gram of tissue, found in each organ containing arelatively high quantity of M₄N. FIG. 1B represents those organscontaining a relatively low quantity of M₄N.

FIG. 2 shows systemic tissue distribution profile of M₄N at various timepoints. Mice were injected with 100 μCi of ³H— M₄N and 60 mM ofunlabeled M₄N. Organs and blood were harvested and weighed at 4, 6, 18hours and 6 days post-injection and the M₄N was extracted. The tritiumcontent of the organ extracts were measured, and the quantity of M₄N ineach organ was calculated based on the specific activity of theinoculum.

FIG. 3 shows the body weights of mice during long-term oral feeding ofM₄N, indicating no apparent toxicity. Male and female mice werecontinually fed food balls weighing 9 g and containing 280 mg of M₄N for14 weeks. On average, mice consumed 93.3 mg of M₄N per day. Control micewere fed food balls containing no M₄N. Body weights were recordedperiodically.

FIG. 4 shows that systemic treatment with M₄N inhibits the in vivogrowth of human tumor xenografts. Athymic nude mice were implanted s.c.in each flank with MCF-7 breast adenocarcinoma cells, Hep3Bhepatocellular carcinoma cells, HT-29 colorectal carcinoma cells, andLNCaP prostate carcinoma cells. When tumors attained a mean diameter of7-8 mm, mice received for three weeks a single daily i.p. injectioncontaining 2 mg of M₄N dissolved in 100 μL Cremaphor-ethanol basedsolvent. Control mice received vehicle only. Tumors were measured in twoperpendicular dimensions (L and W) once every seven days, and tumorvolumes were calculated according to the formula: V=(L×W/2)³×π/6.

FIG. 5 shows the serum concentration of M₄N in dogs given differentdoses of M₄N at different time points.

FIG. 6 is a schematic representation of examples of different modes ofdelivery of the NDGA derivatives to the brain for treatment of braintumors. M₄N represents a hydrophilic NDGA and G₄N represents alipophilic NDGA. OD represents osmotic disruption of blood brainbarrier. SC represents subcutaneous administration. IP representsintraperitoneal administration. IM represents intramuscularadministration.

FIG. 7 is a schematic representation of examples of different modes ofdelivery of the NDGA derivatives to tissues other than the brain for thetreatment of tumors. M₄N represents a hydrophilic NDGA and G₄Nrepresents a lipophilic NDGA. SC represents subcutaneous administration.IP represents intraperitoneal administration. IM representsintramuscular administration.

Table 1. Oral administration of M₄N results in systemic tissuedistribution. (A) Short-term oral feeding of M₄N. Three mice were fed 30mg of M₄N dissolved in castor oil. At 2, 4, and 8 hours post-feeding,tissues were removed and weighed. The quantity of M₄N present in tissueswas then quantitated by HPLC. (B) Long-term oral feeding of M₄N. Micewere continually fed food balls weighing 9 g and containing 280 mg ofM₄N for 14 weeks. On average, mice consumed 93.3 mg of M₄N per day. Thequantity of M₄N present in tissues was quantitated by HPLC.

Table 2. Systemic treatment with M₄N inhibits the in vivo growth ofhuman tumor xenografts. Athymic nude mice were implanted s.c. in eachflank with MCF-7 breast adenocarcinoma cells, Hep3B hepatocellularcarcinoma cells, HT-29 colorectal carcinoma cells, and LNCaP prostatecarcinoma cells. When tumors attained a mean diameter of 7-8 mm, micereceived for three weeks a single daily i.p. injection containing 2 mgof M₄N dissolved in 100 μL Cremaphor-ethanol based solvent. Control micereceived vehicle only. Tumors were measured in two perpendiculardimensions (L and W) once every seven days, and tumor volumes werecalculated according to the formula: V=(L×W/2)³×π/6.

Table 3. Tumor size change for all tumors following 21 days oftreatment. Athymic nude mice were implanted s.c. in each flank withMCF-7 breast adenocarcinoma cells, Hep3B hepatocellular carcinoma cells,HT-29 colorectal carcinoma cells, and LNCaP prostate carcinoma cells.When tumors attained a mean diameter of 7-8 mm, mice received for threeweeks a single daily i.p. injection containing 2 mg of M₄N dissolved in100 μL Cremaphor-ethanol based solvent. Control mice received vehicleonly. Tumors were measured in two perpendicular dimensions (L and W)once every seven days, and tumor volumes were calculated according tothe formula: V=(L×W/2)³×π/6.

DETAILED DESCRIPTION OF THE INVENTION

The inventors herein have discovered that catecholic butanes of theformula I:

where R₁ and R₂ are independently —H, a lower alkyl, a lower acyl, analkylene or an unsubstituted or substituted amino acid residue or saltthereof; R₃, R₄, R₅, R₆, R₁₀, R₁₁, R₁₂ and R₁₃ are independently —H or alower alkyl; and R₇, R₈ and R₉ are independently —H, —OH, a loweralkoxy, a lower acyloxy, or any two adjacent groups together may be analkyene dioxy, or an unsubstituted or substituted amino acid residue orsalt thereof are useful for the treatment of proliferative diseases suchas cancer, when applied other than by direct injection into the tumor ortopically onto the situs of the tumor. Such catecholic butanes can becombined with pharmaceutically acceptable carrier or excipient toproduce pharmaceutical compositions that can be formulated for differentroutes of delivery.

In another embodiment of the invention, the catecholic butane has theformula above where R₁ and R₂ are independently —H, a lower alkyl, alower acyl, or an unsubstituted or substituted amino acid residue orsalt thereof; R₃, R₄, are independently a lower alkyl; R₅, R₆, R₁₀, R₁₁,R₁₂ and R₁₃ are independently —H; and R₇, R₈ and R₉ are independently—H, —OH, a lower alkoxy, a lower acyloxy, or an unsubstituted orsubstituted amino acid residue or salt thereof.

In a further embodiment of the invention, the pharmaceutical compositionhas the above formula where R₁ and R₂ are independently —H, a loweralkyl, a lower acyl, or an unsubstituted or substituted amino acidresidue or salt thereof; R₃, R₄, are independently a lower alkyl; R₅,R₆, R₇, R₁₀, R₁₁, R₁₂ and R₁₃ are independently —H; and R₈ and R₉ areindependently —OH, a lower alkoxy, lower acyloxy, or an unsubstituted orsubstituted amino acid residue or salt thereof.

In a further embodiment of the invention, the pharmaceutical compositionhas the formula above where R₁ and R₂ are independently —CH₃ or—(C═O)CH₂N(CH₃)₂ or a salt thereof.

In another embodiment of the invention, the pharmaceutical compositionhas the formula above where R₈ and R₉ are independently —OCH₃ or—O(C═O)CH₂N(CH₃)₂ or a salt thereof.

In a further embodiment of the invention, the pharmaceutical compositionhas the formula above where R₁ and R₂ are independently —CH₃,—(C═O)CH₂N(CH₃)₂ or —(C═O)CH₂N⁺H(CH₃)₂.Cl⁻ and R₈ and R₉ areindependently —OCH₃, —O(C═O)CH₂N(CH₃)₂ or —O(C═O)CH₂N⁺H(CH₃)₂.Cl⁻.

In yet another embodiment of the invention, the pharmaceuticalcomposition has the formula above where R₁ and R₂ are independently —Hor —CH₃ and R₈ and R₉ are independently —OH or —OCH₃, provided that thecatecholic butane is not NDGA.

In a different embodiment of the invention, the pharmaceuticalcomposition has the formula as above where R₁ and R₂ are independently—CH₃ and R₈ and R₉ are independently —OCH₃.

In yet another embodiment of the invention, the catecholic butane isNDGA. In an alternative embodiment, the catecholic butane is other thanNDGA, namely, a NDGA derivative with the following formula II:

The present inventors have surprisingly discovered that a compositioncontaining a substantially pure preparation of at least one NDGAderivative is effective for the treatment of proliferative diseases suchas tumors, when such composition is administered via a route other thanthe direct injection into the affected or target tissues, and other thanby topical application onto the affected tissue. The NDGA derivativesherein have a formula II as set forth above, where R₁, R₂, R₃ and R₄independently represent —OH, a lower alkoxy, for example, —OCH₃, a loweracyloxy, for example, —O(C═O)CH₃, or unsubstituted or substituted aminoacid residue or salt thereof but are not each —OH simultaneously; andR₅, R₆ independently represent —H or an alkyl such as a lower alkyl, forexample, —CH₃ or —CH₂CH₃. In one embodiment, R₅ and R₆ can both be —H,—CH₃ or —CH₂CH₃.

The present catecholic butane, including the NDGA Compounds, in asuitable formulation, can be safely administered to one or more subjectsin need of such treatment by intranasal delivery. Optionally, suchcatecholic butanes or NDGA Compounds can be administered by inhalation.Further optionally, such catecholic butanes or NDGA Compounds can beadministered orally, such as by mixing with food, for example, orbuccally, or intraocularly. Additionally, the catecholic butanes or NDGACompounds can be administered as an oral rinse, for example, in arinse-and-spit treatment one or more times a day.

Moreover, the catecholic butanes or NDGA Compounds formulated inliposomal formulations, nanoparticle formulations, or micellarformulations can additionally be safely administered systemically, suchas intravenously, such as by injection into the central vein forexample, or intraperitoneally, interstitially, subcutaneously,transdermally, intramuscularly, intra-arterially, intra-cranially, orintra-ventricularly.

Furthermore, the catecholic butanes or NDGA Compounds can be formulatedin liposomal formulations, nanoparticles formulations, or micellarformulations, or any formulation embedded in a biodegradable polymer,for administration into a subject, such as one in need of suchtreatment. Implantation into the brain, for example, can be used fortreatment of brain tumors.

In one embodiment of the invention, the route of administration forpurposes herein is other than by parenteral administration, whereparenteral administration herein means intravenous, intra-arterial,intramuscular, subcutaneous, transdermal and intraperitonealadministration.

The present invention further features a pharmaceutical compositioncontaining catecholic butanes or NDGA Compounds for treatment ofproliferative diseases such as tumors where the composition isformulated for delivery or administration as described above such as,for example, in the form of a tablet, a liquid that is eitherhydrophilic or hydrophobic, a powder such as one resulting fromlyophilization, an aerosol, or in the form of an aqueous water solublecomposition, a hydrophobic composition, a liposomal composition, amicellar composition, such as that based on Tween 80 or diblockcopolymers, a nanoparticle composition, a polymer composition, acyclodextrin complex composition, emulsions, lipid based nanoparticlestermed “lipocores.”

The present invention further features a method of producing thepharmaceutical composition of the present invention, the methodinvolving making or providing the catecholic butanes or NDGA Compoundsin a substantially purified form, combining the composition with apharmaceutically acceptable carrier or excipient, and formulating thecomposition in a manner that is compatible with the mode of desiredadministration.

In a further aspect of the present invention, there is provided a methodof treating tumor as above, where the tumor is selected from the groupconsisting of lung, prostate, breast, colon, liver, kidney, ovarian,cervical, skin, pancreas, brain, leukemias, lymphomas, gastrointestinaltumor such as stomach, soft tissue sarcomas and the like.

The present invention still additionally provides for kits comprisingcompositions or formulations as above for the treatment of proliferativediseases such as tumors where the compositions are formulated fordelivery as above, including but not limited to intranasaladministration, inhalation, oral administration, intravenousadministration, intraperitoneal administration and other parenteraladministration, or as an oral rinse, or the like, and instructions forsuch administration.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The present invention may bebetter understood in light of the particular meanings as follows.

The term “active agent,” “compound,” and “drug” herein refers to one ormore catecholic butanes, including NDGA and NDGA derivatives.

The term “alkylene dioxy” as used herein refers to methylene (orsubstituted methylene) dioxy or ethylene (or substituted ethylene)dioxy.

The term “unsubstituted or substituted amino acid residue or saltthereof” in reference to one of the R groups in the formula for thecatecholic butane herein is an amino acid residue or a substituted aminoacid residue or salt of an amino acid residue or salt of a substitutedamino acid residue including but not limited to: alanine, arginine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine,5-hydroxylysine, 4-hydroxyproline, thyroxine, 3-methylhistidine,ε-N-methyllysine, ε-N,N,N-trimethyllysine, aminoadipic acid,γ-caroxyglutamic acid, phosphoserine, phosphothreonine, phosphotyrosine,N-methylarginine, N-acetyllysine, and an N,N-dimethyl-substituted aminoacid residue; or a salt thereof, such as a chloride salt.

The term “lower alkyl” means C₁-C₆ alkyl.

The term “lower acyl” means C₁-C₆ acyl.

The term “NDGA Compound” refers to NDGA and/or its derivatives, singlyor collectively.

The term “NDGA derivative” refers to a derivative of NDGA each havingthe formula II:

wherein R₁, R₂, R₃ and R₄ are independently —OH, lower alkoxy, loweracyloxy, or an unsubstituted or substituted amino acid residue or saltthereof but are not each —OH simultaneously; and R₅, R₆ areindependently —H or an alkyl such as a lower alkyl. The term includes,for example, a compound in which R₁, R₂, R₃ and R₄ are each —OCH₃, orare each —O(C═O)CH₃; and R₅, R₆ are each —H or each a lower alkyl. Inone embodiment of the invention, R₅, R₆ are each —CH₃ or —CH₂CH₃.

A “substantially purified” compound in reference to the catecholicbutanes or NDGA Compounds herein is one that is substantially free ofcompounds that are not the catecholic butane or NDGA Compounds of thepresent invention (hereafter, “non-NDGA materials”). By substantiallyfree is meant at least 50%, preferably at least 70%, more preferably atleast 80%, and even more preferably at least 90% free of non-NDGAmaterials.

The “buffer” suitable for use herein includes any buffer conventional inthe art, such as, for example, Tris, phosphate, imidazole, andbicarbonate.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a condition or disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a condition ordisease and/or adverse affect attributable to the condition or disease.“Treatment,” thus, for example, covers any treatment of a condition ordisease in a mammal, particularly in a human, and includes: (a)preventing the condition or disease from occurring in a subject whichmay be predisposed to the condition or disease but has not yet beendiagnosed as having it; (b) inhibiting the condition or disease, suchas, arresting its development; and (c) relieving, alleviating orameliorating the condition or disease, such as, for example, causingregression of the condition or disease.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any conventional type. A “pharmaceuticallyacceptable carrier” is non-toxic to recipients at the dosages andconcentrations employed, and is compatible with other ingredients of theformulation. For example, the carrier for a formulation containing thepresent catecholic butane or NDGA Compounds preferably does not includeoxidizing agents and other compounds that are known to be deleterious tosuch. Suitable carriers include, but are not limited to, water,dextrose, glycerol, saline, ethanol, buffer, dimethyl sulfoxide,Cremaphor EL, and combinations thereof. The carrier may containadditional agents such as wetting or emulsifying agents, or pH bufferingagents. Other materials such as anti-oxidants, humectants, viscositystabilizers, and similar agents may be added as necessary.

Pharmaceutically acceptable salts herein include the acid addition salts(formed with the free amino groups of the polypeptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, mandelic, oxalic, andtartaric. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, and histidine.

The term “pharmaceutically acceptable excipient,” includes vehicles,adjuvants, or diluents or other auxiliary substances, such as thoseconventional in the art, which are readily available to the public. Forexample, pharmaceutically acceptable auxiliary substances include pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like.

The terms “subject,” “host,” and “patient,” are used interchangeablyherein to refer to an animal being treated with the presentcompositions, including, but not limited to, simians, humans, felines,canines, equines, bovines, porcines, ovines, caprines, mammalian farmanimals, mammalian sport animals, and mammalian pets.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

All publications mentioned herein, including patents, patentapplications, and journal articles are incorporated herein by referencein their entireties including the references cited therein, which arealso incorporated herein by reference.

It must be noted that as used herein, the singular forms “a”, “an”, and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a compound” includes aplurality of such compounds and reference to “the NDGA Compound”includes reference to one or more NDGA Compounds and equivalents thereofknown to those skilled in the art.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The invention described below is given by way of example only and is notto be interpreted in any way as limiting the invention.

Preparation of Catecholic Butanes

The catecholic butanes of the present invention can be prepared by anyconventional methodologies. For example, such compounds can be made asdescribed in U.S. Pat. No. 5,008,294.

Preparation of the NDGA Compounds

The NDGA Compounds and formulations thereof can be made by any processconventional in the art. For example, the NDGA Compounds can be made asdescribed in, U.S. Pat. No. 5,008,294 (Jordan et al., issued Apr. 16,1991); U.S. Pat. No. 6,291,524 (Huang et al., issued Sep. 18, 2001);Hwu, J. R. et al. (1998); or McDonald, R. W. et al. (2001).

In one embodiment of the present invention, an NDGA Compound,tetra-O-methyl NDGA, also known asmeso-1,4-bis(3,4-dimethoxyphenyl)-2,3-dimethylbutane, or M₄N is made asfollows: a solution is made containing NDGA and potassium hydroxide inmethanol in a reaction flask. Dimethyl sulfate is then added to thereaction flask and the reaction is allowed to proceed. The reaction isfinally quenched with water, causing the product to precipitate. Theprecipitate is isolated by filtration and dried in a vacuum oven. Thecompound is then dissolved in a solution of methylene chloride andtoluene and subsequently purified through an alumina column. Thesolvents are removed by rotary evaporation and the solid is resuspendedin isopropanol and isolated by filtration. The filter cake is dried in avacuum oven. The resulting tetra-O-methyl NDGA (M₄N) is crystallized byrefluxing the filter cake in isopropanol and re-isolating the crystalsby filtration.

In some embodiments of the present invention, certain NDGA Compounds ofthe present invention, such as G₄N, also known asmeso-1,4-bis[3,4-(dimethylaminoacetoxy)phenyl]-(2R,3S)-dimethylbutane ortetra-dimethylglycinyl NDGA, or a hydrochloride salt thereof and similarcompounds having amino acid substituents, can also be prepared accordingto conventional methods, as described in, for example, U.S. Pat. No.6,417,234.

Compositions

The present invention further provides compositions, includingpharmaceutical compositions, comprising the catecholic butanes includingthe NDGA Compounds and pharmaceutically acceptable carriers orexcipients. These compositions may include a buffer, which is selectedaccording to the desired use of the catecholic butanes or NDGACompounds, and may also include other substances appropriate for theintended use. Those skilled in the art can readily select an appropriatebuffer, a wide variety of which are known in the art, suitable for anintended use. In some instances, the composition can comprise apharmaceutically acceptable excipient, a variety of which are known inthe art. Pharmaceutically acceptable excipients suitable for use hereinare described in a variety of publications, including, for example, A.Gennaro (1995); Ansel, H. C. et al. (1999); and Kibbe, A. H. (2000).

The compositions herein are formulated in accordance to the mode ofpotential administration. Thus, if the composition is intended to beadministered intranasally or by inhalation, for example, the compositionmay be a converted to a powder or aerosol form, as conventional in theart, for such purposes. Other formulations, such as for oral orparenteral delivery, are also used as conventional in the art.

Compositions for administration herein may form solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders.

Therapeutic Methods

The catecholic butanes, including the NDGA Compound compositions of thesubject invention find use as therapeutic agents in situations where onewishes to provide a treatment to a subject who has a proliferativedisease such as a malignant, premalignant or benign tumor and where onewishes to provide treatment to viral diseases such as HIV, HPV or HSV.

A variety of animal hosts are treatable according to the subjectmethods, including human and non-human animals. Generally such hosts are“mammals” or “mammalian,” where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., dogs and cats), rodentia (e.g., guinea pigs, and rats),and other mammals, including cattle, goats, horses, sheep, rabbits,pigs, and primates (e.g., humans, chimpanzees, and monkeys). In manyembodiments, the hosts will be humans. Animal models are of interest forexperimental investigations, such as providing a model for treatment ofhuman disease. Further, the present invention is applicable toveterinary care as well.

Moreover, the compounds of the present invention can be used to treat avariety of tumors and cancers, including, without limitation, acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, anal cancer, astrocytoma, bile duct cancer, bladder cancer,bone cancer osteosarcoma/malignant fibrous histiocytoma, brain stemglioma, brain tumor ependymoma, brain tumor medulloblastoma, breastcancer, carcinoid tumor gastrointestinal, carcinoma adrenocortical,carcinoma islet cell, cervical cancer, chronic lymphocytic leukemia,chronic myelogenous leukemia, clear cell sarcoma of tendon sheaths,colon cancer, cutaneous T-cell lymphoma, endometrial cancer, epithelialcancer ovarian, esophageal cancer, Ewing's family of tumors,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, eye cancer retinoblastoma, gallbladder cancer,gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ celltumor extragonadal, germ cell tumor, ovarian tumor, gestationaltrophoblastic tumor, glioma, hairy cell leukemia, hepatocellular (liver)cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma,islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, laryngealcancer, leukemia acute lymphoblastic cancer, leukemia acute myeloidcancer, leukemia chronic lymphocytic cancer, leukemia chronicmyelogenous cancer, leukemia hairy cell cancer, liver cancer, lungcancer non-small cell, lung cancer small cell, male breast cancer,malignant mesothelioma, medulloblastoma, melanoma, merkel cellcarcinoma, multiple endocrine neoplasia syndrome, mycosis fungoides,myeloma multiple, nasal cavity, paranasal and sinus cancer,nasopharyngeal cancer, neuroblastoma, oral cavity and lip cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian epithelial cancer, ovarian germ cell tumor, pancreaticcancer, parathyroid cancer, penile cancer, pheochromocytoma, pineal andsupratentorial primitive neuroectodermal tumors, pituitary tumor,pleuropulmonary blastoma, prostate cancer, rectal cancer, renal, pelvisand ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, sarcoma soft tissue adult, Sezary syndrome, skincancer, small intestine cancer, stomach (gastric) cancer, testicularcancer, thymoma, thyroid cancer, urethral cancer, uterine cancerendometrial, vaginal cancer, vulvar cancer, Waldenström'smacroglobulinemia, and Wilms' tumor.

Formulations, Dosages, and Routes of Administration

As mentioned above, an effective amount of the active agent isadministered to the host, where “effective amount” means a dosagesufficient to produce a desired result. In some embodiments, the desiredresult is at least a reduction or inhibition of tumor growth as comparedto a control.

Typically, the compositions of the instant invention will contain fromless than about 1% up to about 99% of the active ingredient, that is,the catecholic butanes including the NDGA Compounds herein; optionally,the instant invention will contain about 5% to about 90% of the activeingredient. The appropriate dose to be administered depends on thesubject to be treated, such as the general health of the subject, theage of the subject, the state of the disease or condition, the weight ofthe subject, the size of the tumor, for example. Generally, betweenabout 0.1 mg and about 500 mg or less may be administered to a child andbetween about 0.1 mg and about 5 grams or less may be administered to anadult. The active agent can be administered in a single or, moretypically, multiple doses. Preferred dosages for a given agent arereadily determinable by those of skill in the art by a variety of means.Other effective dosages can be readily determined by one of ordinaryskill in the art through routine trials establishing dose responsecurves. The amount of agent will, of course, vary depending upon theparticular agent used.

The frequency of administration of the active agent, as with the doses,will be determined by the care giver based on age, weight, diseasestatus, health status and patient responsiveness. Thus, the agents maybe administered one or more times daily, weekly, monthly or asappropriate as conventionally determined. The agents may be administeredintermittently, such as for a period of days, weeks or months, then notagain until some time has passed, such as 3 or 6 months, and thenadministered again for a period of days, weeks, or months.

The catecholic butanes or active agents of the present invention can beincorporated into a variety of formulations for therapeuticadministration. More particularly, the catecholic butanes of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, aerosols,liposomes, nanoparticles, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

As such, administration of the active agents can be achieved in variousways, such as oral, buccal, rectal, intranasal, intravenous,intra-arterial, intra-tracheal, intraventricular, intracranial,interstitial, transdermal, etc., or by inhalation or implantation.

In particular, nanoparticle, micelle and liposomal preparation can beadministered systemically, including parenterally and intranasally, aswell as interstitially, orally, topically, transdermally, via inhalationor implantation, such as for drug targeting, enhancement of drugbioavailability and protection of drug bioactivity and stability.Nanoparticle bound drugs herein are expected to achieve prolonged drugretention in tumors.

In pharmaceutical dosage forms, the active agents may be administered inthe form of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the active agents can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents. For oral rinses,the preparations can be made in a manner conventional in the art, suchas described in, for example, Epstein, J. B. et al. (2002) and Pitten,F. et al. (2003).

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are conventional in the art. Suitable excipientvehicles are, for example, water, saline, dextrose, glycerol, ethanol,or the like, and combinations thereof. In addition, if desired, thevehicle may contain minor amounts of auxiliary substances such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents or emulsifying agents. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in theart. See, e.g., Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 17th edition, 1985. The composition or formulationto be administered will, in any event, contain a quantity of the agentadequate to achieve the desired state in the subject being treated.

The active agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or non-aqueoussolvent, such as vegetable or other similar oils, including corn oil,castor oil, synthetic aliphatic acid glycerides, esters of higheraliphatic acids or propylene glycol; and if desired, with conventionaladditives such as solubilizers, isotonic agents, suspending agents,emulsifying agents, stabilizers and preservatives.

The active agents can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the active agents can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The compounds of the present invention can be administeredrectally via a suppository. The suppository can include vehicles such ascocoa butter, carbowaxes and polyethylene glycols, which melt at bodytemperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Kits with multiple or unit doses of the active agent, are included inthe present invention. In such kits, in addition to the containerscontaining the multiple or unit doses of the compositions containing theNDGA derivatives will be an informational package insert withinstructions describing the use and attendant benefits of the drugs intreating pathological condition of interest.

Tumors which may be treated using the methods of the instant inventioninclude carcinomas, e.g. colon, rectum, prostate, breast, melanoma,ductal, endometrial, stomach, pancreatic, mesothelioma, dysplastic oralmucosa, invasive oral tumor, non-small cell lung carcinoma (“NSCL”),transitional and squamous cell urinary carcinoma, etc.; neurologicalmalignancies, e.g. neuroblastoma, glioblastoma, astrocytoma, gliomas,etc.; hematological malignancies, e.g. childhood acute leukaemia,non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignantcutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma,lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia,bullous pemphigoid, discoid lupus erythematosus, lichen planus, etc.;gynecological tumors, e.g., cervical and ovarian; testicular tumors;liver tumors including hepatocellular carcinoma (“HCC”) and tumor of thebiliary duct; multiple myelomas; tumors of the esophageal tract; otherlung tumors including small cell and clear cell; Hodgkin's lymphomas;sarcomas in different organs; as well as those mentioned above; and thelike.

Preparation of Nanoparticles (“NP”)

The present invention includes formulations of catecholic butanes,including NDGA Compounds, in a NP preparation. A number of different NPformulations suitable for use herein can be made depending on the methodof delivery. The NP formulation can differ based on the drug releaseprofile desired, by controlling the molecular weight, the copolymerratio, the drug loading, the microparticle size and porosity and thefabrication conditions. The NP formulations can also differ on the basisof polymers, stabilizers, and surfactants used in the productionprocess. Different excipients may also have different effects on druguptake, drug distribution throughout the body and persistence of thedrug in plasma. A person having skills conventional in the art will beable to determine the desired properties or characteristics, andaccordingly determine the appropriate NP formulation to use.

The polymeric matrix of the NP must meet the criteria ofbiocompatibility, bioavailability, mechanical strength and ease ofprocessing. The best known polymers for this purpose is thebiodegradable poly(lactide-co-glycolide)s (“PLGAs”).

NP herein can be made by any process conventional in the art. In oneembodiment, the NP can be made as described in, for example, Lockman, P.R., et al. (2002). The types of manufacturing process include, forexample, emulsion polymerization, interfacial polymerization,desolvation evaporation and solvent deposition.

In the emulsion polymerization process of making the NP herein, thepolymerization process consists of building a chain of polymers from asingle monomer unit, as described in, for example, Kreuter, J. (1994).Polymerization occurs spontaneously at room temperature after initiationby either free radical or ion formation, such as by use of high-energyradiation, UV light, or hydroxyl ions. Once polymerization is completethe solution is filtered and neutralized. The polymers form micelles anddroplets consisting of from about 100 to 10⁷ polymer molecules.Surfactants and stabilizers are generally not need in this process.Also, this process can be accomplished in an organic phase rather thanan aqueous phase.

The NP herein can also be made by an interfacial polymerization processas described in, for example, Khouri, A. I., et al. (1986). In thisprocess, monomers are used to create the polymer and polymerizationoccurs when an aqueous and organic phase are brought together byhomogenization, emulsification, or micro-fluidization under high-torquemechanical stirring. For example, polyalkylcyanoacrylate nanocapsulescontaining the catecholic butanes, such as the NDGA Compounds, can bemade by combining the lipophilic NDGA Compounds and the monomer in anorganic phase, dissolving the combination in oil, and slowly adding themixture through a small tube to an aqueous phase with constant stirring.The monomer can then spontaneously form 200-300 nm capsules by anionicpolymerization. A variation of this process involves adding a solventmixture of benzyl benzoate, acetone, and phospholipids to the organicphase containing the monomer and the drug, as described in, for example,Fessi, H., et al. (1989). This creates a formulation in which the drugis encapsulated and protected against degradation until it reaches thetarget tissue.

Macromolecules such as albumin and gelatin can be used in oildenaturation and desolvation processes in the production of NPs. In theoil emulsion denaturation process, large macromolecules are trapped inan organic phase by homogenization. Once trapped, the macromolecule isslowly introduced to an aqueous phase undergoing constant stirring. Thenanoparticles formed by the introduction of the two immiscible phasescan then be hardened by crosslinking, such as with an aldehyde or byheat denaturation.

Alternatively, macromolecules can form NPs by “desolvation.” In thedesolvation process, macromolecules are dissolved in a solvent in whichthe macromolecules reside in a swollen, coiled configuration. Theswollen macromolecule is then induced to coil tightly by changing theenvironment, such as pH, charge, or by use of a desolvating agent suchas ethanol. The macromolecule may then be fixed and hardened bycrosslinking to an aldehyde. The NDGA Compounds can be adsorbed or boundto the macromolecule before crosslinking such that the derivativesbecome entrapped in the newly formed particle.

Solid lipid NP can be created by high-pressure homogenization. Solidlipid NPs have the advantage that they can be sterilized and autoclavedand possess a solid matrix that provides a controlled release.

The present invention further includes NP with different methods of drugloading. The NP can be solid colloidal NP with homogeneous dispersion ofthe drug therein. The NP can be solid NP with the drug associated on theexterior of the NP, such as by adsorption. The NP can be a nanocapsulewith the drug entrapped therein. The NP can further be solid colloidalNP with homogeneous dispersion of the drug therein together with a cellsurface ligand for targeting delivery to the appropriate tissue.

The size of the NPs may be relevant to their effectiveness for a givenmode of delivery. The NPs typically ranges from about 10 nm to about1000 nm; optionally, the NPs can range from about 30 to about 800 nm;further typically, from about 60 to about 270 nm; even furthertypically, from about 80 to about 260 nm; or from about 90 to about 230nm, or from about 100 to about 195. Several factors influence the sizeof the NPs, all of which can be adjusted by a person of ordinary skillin the art, such as, for example, pH of the solution used duringpolymerization, amount of initiation triggers (such as heat orradiation, etc.) and the concentration of the monomer unit. Sizing ofthe NPs can be performed by photon correlation spectroscopy using lightscattering.

The NPs herein, such as polysaccharide NPs or albumin NPs, mayoptionally be coated with a lipid coating. For example, polysaccharideNPs can be crosslinked with phosphate (anionic) and quarternary ammonium(cationic) ligands, with or without a lipid bilayer, such as onecontaining dipalmitoyl phosphatidyl choline and cholesterol coating.Other polymer/stabilizer include, but is not limited to: soybean oil;maltodextrin; polybutylcyanoacrylate; butylcayanoacrylate/dextran 70kDa, Polysorbate-85; polybutylcyanoacrylate/dextran 70 kDa,polysorbate-85; stearic acid; poly-methylmethylacrylate.

The NP preparations containing the catecholic butanes, such as the NDGACompounds, such as by adsorption to the NPs, can be administeredintravenously for treatment of tumors, for example, in the brain, heartand reticuloendothelial cell (“RES”) containing organs, such as liver,spleen and bone marrow. To avoid undesirable uptake of these NPpreparations by the reticuloendothelial cells, the NPs may be coatedwith a surfactant or manufactured with a magnetically responsivematerial.

Thus, optionally, a surfactant may be used in conjunction with the NP.For example, polybutylcyanoacrylate NPs can be used with adextran-70,000 stabilizer and Polysorbate-80 as a surfactant. Othersurfactants include, but not limited to: Polysorbate-20, 40, or 60;Poloxamer 188; lipid coating-dipalmitoyl phosphatidylcholine; Epikuron200; Poloxamer 338; Polaxamine 908; Polaxamer 407. For example,Polyaxamine 908 may be used as a surfactant to decrease uptake of NPsinto the RES of the liver, spleen, lungs, and bone marrow.

The magnetically responsive material can be magnetite (Fe₃O₄) which canbe incorporated into the composition for making the NP. Thesemagnetically responsive NPs can be externally guided by a magnet.

In another embodiment, the NPs herein can be made as described in Mu, L.and Feng, S. S. (2003), using a blend of poly(lactide-co-glycolide)s(“PLGAs”) and d-α-tocopheryl polyethylene glycol 1000 succinate (vitaminE TPGS or TPGS). The latter can also act as an emulsifier, in additionto being a matrix material.

Preparation of Micelle Forming Carriers

The present invention includes catecholic butanes, including the NDGACompounds, formulated in micelle forming carriers, where the micellesare produced by processes conventional in the art. Examples of such aredescribed in, for example, Liggins, R. T. and Burt, H. M. (2002); Zhang,X. et al. (1996); and Churchill, J. R. and Hutchinson, F. G. (1988). Inone such method, polyether-polyester block copolymers, which areamphipathic polymers having hydrophilic (polyether) and hydrophobic(polyester) segments, are used as micelle forming carriers.

Another type of micelles is, for example, that formed by the AB-typeblock copolymers having both hydrophilic and hydrophobic segments, whichare known to form micellar structures in aqueous media due to theiramphiphilic character, as described in, for example, Tuzar, Z. andKratochvil, P. (1976); and Wilhelm, M. et al. (1991). These polymericmicelles are able to maintain satisfactory aqueous stabilityirrespective of the high content of hydrophobic drug incorporated withinthe micelle inner core. These micelles, in the range of approximately<200 nm in size, are effective in reducing non-selective RES scavengingand shows enhanced permeability and retention at solid tumor sites. Thischaracteristic allows for the accumulation of anti-cancer drug, such asthe NDGA derivatives, to accumulate at the cancer site.

Further, for example, poly(D,L-lactide)-b-methoxypolyethylene glycol(MePEG:PDLLA) diblock copolymers can be made using MePEG 1900 and 5000.The reaction can be allowed to proceed for 3 hr at 160° C., usingstannous octoate (0.25%) as a catalyst. However, a temperature as low as130° C. can be used if the reaction is allowed to proceed for about 6hr, or a temperature as height as 190° C. can be used if the reaction iscarried out for only about 2 hr.

In one embodiment, N-isopropylacrylamide (“IPAAm”) (Kohjin, Tokyo,Japan) and dimethylacrylamide (“DMAAm”) (Wako Pure Chemicals, Tokyo,Japan) can be used to make hydroxyl-terminated poly(IPAAm-co-DMAAm) in aradical polymerization process, using the method of Kohori, F. et al.(1998). The obtained copolymer can be dissolved in cold water andfiltered through two ultrafiltration membranes with a 10,000 and 20,000molecular weight cut-off. The polymer solution is first filtered througha 20,000 molecular weight cut-off membrane. Then the filtrate wasfiltered again through a 10,000 molecular weight cut-off membrane. Threemolecular weight fractions can be obtained as a result, a low molecularweight, a middle molecular weight, and a high molecular weight fraction.A block copolymer can then be synthesized by a ring openingpolymerization of D,L-lactide from the terminal hydroxyl group of thepoly(IPAAm-co-DMAAm) of the middle molecular weight fraction. Theresulting poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) copolymer can bepurified as described in Kohori, F., et al. (1999).

The catecholic butanes, such as the NDGA Compounds, can be loaded intothe inner cores of micelles and the micelles prepared simultaneously bya dialysis method. For example, a chloride salt of the NDGA Compoundscan be dissolved in N,N-dimethylacetamide (“DMAC”) and added bytriethylamine (“TEA”). The poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide)block copolymer can be dissolved in DMAC, and distilled water can beadded. The solution of NDGA Compounds and the block copolymer solutioncan be mixed at room temperature, followed by dialysis against distilledwater using a dialysis membrane with 12,000-14,000 molecular weightcut-off (Spectra/Por®2, spectrum Medical Indus., CA. U.S.A.) at 25° C.Poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) micelles incorporating NDGACompounds can be purified by filtration with a 20 nm pore sizedmicrofiltration membrane (ANODISC™, Whatman International), as describedin Kohori, F., et al. (1999).

Preparation of Multivesicular Liposomes Containing NDGA Compounds

Multivesicular liposomes (“MVL”) can be produced by any methodconventional in the art, such as, for example, the double emulsificationprocess as described in Mantripragada, S. (2002). Briefly, in the doubleemulsification process, a “water-in-oil” emulsion is first made bydissolving amphipathic lipids, such as a phospholipid containing atleast one neutral lipid, such as a triglyceride, in one or more volatileorganic solvents, and adding to this lipid component an immiscible firstaqueous component and a hydrophobic catecholic butane, such as ahydrophobic NDGA Compound. The mixture is then emulsified to form awater-in-oil emulsion, and then mixed with a second immiscible aqueouscomponent followed by mechanical mixing to form solvent spherulessuspended in the second aqueous component, forming awater-in-oil-in-water emulsion. The solvent spherules will containmultiple aqueous droplets with the catecholic butane, such as the NDGACompound dissolved in them. The organic solvent is then removed from thespherules, generally by evaporation, by reduced pressure or by passing astream of gas over or through the suspension. When the solvent iscompletely removed, the spherules become MVL, such as DepoFoamparticles. When the neutral lipid is omitted in this process, theconventional multilamellar vesicles or unilamellar vesicles will beformed instead of the MVL.

Formulation of Catecholic Butanes, Such as NDGA Compounds for OralDelivery

Some catecholic butanes, such as NDGA Compounds are water-soluble,hydrophilic compounds, such as G₄N. This invention includes formulationof hydrophilic compounds in a pharmaceutically acceptable carrier orexcipient and delivery of such as oral formulations, such as in the formof an aqueous liquid solution of the compound, or the compounds can belyophilized and delivered as a powder, or made into a tablet, or thecompounds can be encapsulated.

The tablets herein can be enteric coated tablets. The formulationsherein can be sustained release, either slow release or rapid releaseformulations.

The amount of the catecholic butanes, such as NDGA Compounds, to beincluded in the oral formulations can be adjusted depending on thedesired dose to be administered to a subject. Such an adjustment iswithin the skill of persons conventional in the art.

Some catecholic butanes, including some NDGA Compounds, are hydrophobicor lipophilic compounds, such as M₄N. The absorption of lipophiliccompounds in the gut can be improved by using pharmaceuticallyacceptable carriers that can enhance the rate or extent ofsolubilization of the compound into the aqueous intestinal fluid.Lipidic carriers are known in the art, such as, for example, asdescribed in Stuchlik, M. and Zak, S. (2001) The formulations herein canbe delivered as oral liquids or can be encapsulated into various typesof capsules.

The present invention includes, in one embodiment, a formulationcontaining the lipophilic NDGA Compounds that are formulated for oraldelivery by dissolution of such compounds in triacylglycerols, and theformulation is then encapsulated for oral delivery. Triacyglycerols aremolecules with long chain and/or medium chain fatty acids linked to aglycerol molecule. The long chain fatty acids range from about C₁₄ toC₂₄, and can be found in common fat. The medium chain fatty acids rangefrom about C₆ to C₁₂, and can be found in coconut oil or palm kerneloil. Triacylglycerols suitable for use herein include structured lipidsthat contain mixtures of either short-chain or medium chain fatty acidsor both, esterified on the same glycerol molecule.

In another embodiment of the present invention, one or more surfactantscan be added to a mixture of catecholic butanes, including NDGACompounds, and lipidic carrier such that the drug is present in finedroplets of oil/surfactant mix. The surfactants can act to disperse theoily formulation on dilution in the gastrointestinal fluid.

The present invention also includes a formulation for oral delivery ofthe catecholic butanes, including NDGA Compounds, in the form of amicro-emulsion consisting of hydrophilic surfactant and oil. Themicro-emulsion particles can be surfactant micelles containingsolubilized oil and drug.

Also suitable for oral administration are formulations of the catecholicbutanes, including NDGA Compounds, in a solid lipid nanoparticlepreparation. Solid lipid nanoparticles can be prepared in any mannerconventional in the art, such as, for example, as described in Stuchlik,M. and Zak, S. (2001).

In one embodiment, the solid lipid nanoparticle can be prepared in a hothomogenization process by homogenization of melted lipids at elevatedtemperature. In this process, the solid lipid is melted and thecatecholic butane, such as the NDGA Compound, is dissolved in the meltedlipid. A pre-heated dispersion medium is then mixed with the drug-loadedlipid melt, and the combination is mixed with a homogenisator to form acoarse pre-emulsion. High pressure homogenization is then performed at atemperature above the lipids melting point to produce aoil/water-nanoemulsion. The nanoemulsion is cooled down to roomtemperature to form solid lipid nanoparticles.

In another embodiment of the present invention, the solid lipidnanoparticles can be prepared in a cold homogenization process. In thisprocess, the lipid is melted and the catecholic butane, such as the NDGACompound, is dissolved in the melted lipid. The drug-loaded lipid isthen solidified in liquid nitrogen or dry ice. The solid drug-lipid isground in a powder mill to form 50-100 μm particles. The lipid particlesare then dispersed in cold aqueous dispersion medium and homogenized atroom temperature or below to form solid lipid nanoparticles.

The present invention also includes formulation of the lipophiliccatecholic butanes, such as NDGA Compounds, in liposomes or micelles fororal delivery. These formulations can be made in any manner conventionalin the art. Micelles are typically lipid monolayer vesicles in which thehydrophobic drug associates with the hydrophobic regions on themonolayer. Liposomes are typically phospholipids bilayer vesicles. Thelipophilic catecholic butane, such as the lipophilic NDGA Compounds,will typically reside in the center of these vesicles.

Intra-Arterial Administration

The present invention includes formulation of the catecholic butanes, asexemplified by the NDGA Compounds, for intra-arterial administration asis conventional in the art, as described in, for example, Doolittle, N.D. et al. (2000); and Cloughesy, T. F. et al. (1997), with or withoutaccompanying blood brain barrier disruption (“BBBD”), and with orwithout occlusion, such as in hepatic artery chemoemobolization, asdescribed in Drougas, J. G. et al. (1998); and Desai, D. C. et al.(2001). Briefly, where NDGA Compounds are administered intra-arteriallywith occlusion, primary arteries leading to the target site arecatheterized and the NDGA Compounds are applied through a catheter.Embolization of the arteries, in order to retain the NDGA Compounds atthe target site for a longer period, is performed using polyvinylalcohol particles alone or in combination with coils. Intra-arterialdelivery of the NDGA Compounds is limited to water soluble compositions.Water soluble NDGA Compounds, such as G₄N, for example, liposomalformulations of hydrophobic NDGA Compounds, such as M₄N, for example, ornanoparticle formulations of hydrophobic NDGA Compounds are particularlysuited for this type of delivery. The drugs or agents herein can bedissolved in saline prior to intra-arterial injection and such injectionmay be preceded by heparin treatment and sedation. For safest treatmentof brain tumor, preferably, intra-arterial administration is conductedbefore tumor burden becomes excessive.

Osmotic disruption of the blood brain barrier (“BBB”) as conventional inthe art may accompany intra-arterial delivery of the agents herein asdescribed in, for example, Doolittle, N. D. et al. (2000); Sato, S. etal., Acta Neurochir (Wien) 140: 1135-1141; disc 1141-1132 (1998); andBhattacharjee, A. K. et al. Brain Res Protocol 8: 126-131 (2001). Such aprocedure can be used to increase the transfer of drugs into the centralnervous system (“CNS”) preferably just prior to intra-arterial delivery.For such disruption, a catheter is placed into an artery, usually thesuperficial temporal artery, leading to the brain and the BBB isdisrupted with a solution of mannitol. This invasive procedure istypically performed while the patient is under general anesthesia. Suchtreatment may require prior hydration and administration ofanticonvulsants and/or atropine.

Formulation of NDGA Compounds for Intranasal Delivery

The present invention includes formulations of catecholic butanes, asexemplified by the NDGA Compounds, for intranasal delivery andintranasal delivery thereof. Intransal delivery may advantageously buildup a higher concentration of the active agents in the brain than can beachieved by intravenous administration. Also, this mode of deliveryavoids the problem of first pass metabolism in the liver and gut of thesubject receiving the drug.

The amount of the active agents that can be absorbed partly depends onthe solubility of the drug in the mucus, a composition that consists ofabout 95% water solution of serum proteins, glycoproteins, lipids andelectrolytes. Generally, as lipophilicity of the active agents hereinincreases, the drug concentration in the CSF also increases. See, forexample, Minn, A. et al. (2002).

The hydrophilic NDGA Compounds can be dissolved in a pharmaceuticallyacceptable carrier such as saline, phosphate buffer, or phosphatebuffered saline. In one embodiment, a 0.05 M phosphate buffer at pH 7.4can be used as the carrier, as described in, for example, Kao, H. D., etal. (2000).

Intranasal delivery of the present agents may be optimized by adjustingthe position of the subject when administering the agents. For example,the head of the patient may be variously positioned upright-90°,supine-90°, supine-45°, or supine-70°, to obtain maximal effect.

The carrier of the composition of NDGA Compounds may be any materialthat is pharmaceutically acceptable and compatible with the activeagents of the composition. Where the carrier is a liquid, it can behypotonic or isotonic with nasal fluids and within the pH of about 4.5to about 7.5. Where the carrier is in powdered form it is also within anacceptable pH range.

The carrier composition for intranasal delivery may optionally containlipophilic substances that may enhance absorption of the active agentsacross the nasal membrane and into the brain via the olfactory neuralpathway. Examples of such lipophilic substances include, but are notlimited to, gangliosides and phosphatidylserine. One or severallipophilic adjuvants may be included in the composition, such as, in theform of micelles.

The pharmaceutical composition of active agents for intranasal deliveryto a subject for treatment of tumor and other proliferative diseases,disorders, or conditions herein can be formulated in the mannerconventional in the art as described in, for example, U.S. Pat. No.6,180,603. For example, the composition herein can be formulated as apowder, granules, solution, aerosol, drops, nanoparticles, or liposomes.In addition to the active agents, the composition may containappropriate adjuvants, buffers, preservatives, salts. Solutions such asnose drops may contain anti-oxidants, buffers, and the like.

Delivery by Implantation

The catecholic butanes herein, as exemplified by the NDGA Compounds, maybe delivered to a subject for treatment by surgical implantation into atumor site, with or without surgical excision of the tumor, such as byimplantation of a biodegradable polymer containing the NDGA Compounds.In one embodiment, this method of treatment can be performed, forexample, after surgical resection, such as in the treatment andresection of brain tumor, as described in, Fleming, A. B. and Saltzman,W. M., Pharmacokinetics of the Carmustine Implant, Clin. Pharmacokinet,41: 403-419 (2002). This method of delivery is applicable to not onlybrain tumors but to other tumors as well. This treatment may be combinedwith other conventional therapy besides or in addition to surgery, suchas radiotherapy, chemotherapy or immunotherapy.

Thus, the biodegradable polymer herein can be any polymer or copolymerthat would dissolve in the interstitial fluid, without any toxicity oradverse effect on host tissues. Preferably, the polymer or monomers fromwhich the polymer is synthesized is approved by the Food and DrugAdministration for administration into humans. A copolymer havingmonomers of different dissolution properties is preferred so as tocontrol the dynamics of degradation, such as increasing the proportionof one monomer over the other to control rate of dissolution.

In one embodiment, the polymer is a copolymer of1,3-bis-(p-carboxyphenoxy)propane and sebacic acid [p(CPP:SA)], asdescribed in Fleming A. B. and Saltzman, W. M., Pharmacokinetics of theCarmustine Implant, Clin. Pharmacokinet, 41: 403-419 (2002); and Brem,H. and Gabikian, P. (2001). In another embodiment, the polymer is acopolymer of polyethylene glycol (“PEG”) and sebacic acid, as describedin Fu, J. et al., (2002).

Polymer delivery systems are applicable to delivery of both hydrophobicand hydrophilic NDGA Compounds herein. The NDGA Compounds are combinedwith the biodegradable polymers and surgically implanted at the tumorsite. Some polymer compositions are also usable for intravenous orinhalation therapy herein.

Delivery Through Inhalation

The catecholic butanes herein, as exemplified by the NDGA Compounds, maybe delivered systemically and/or locally by administration to the lungsthrough inhalation. Inhalation delivery of drugs has been well acceptedas a method of achieving high drug concentration in the pulmonarytissues without triggering substantial systemic toxicity, as well as amethod of accomplishing systemic circulation of the drug. The techniquesfor producing such formulations are conventional in the art. Efficacyagainst pulmonary diseases may be seen with either hydrophobic orhydrophilic NDGA Compounds delivered in this manner.

For pulmonary delivery via inhalation, the NDGA Compounds herein may beformulated into dry powders, aqueous solutions, liposomes,nanoparticles, or polymers and administered, for example, as aerosols.Hydrophilic formulations may also be taken up through the alveolarsurfaces and into the bloodstream for systemic applications.

In one embodiment, the polymers containing the active agents herein aremade and used as described in Fu, J. et al. (2002). For example, thepolymers herein can be polymers of sebacic acid and polyethylene glycol(“PEG”), or can be poly(lactic-co-glycolic) acid (“PLGA”), or polymersof polyethyleneimine (“PEI”) and poly-L-lysine (“PLL”).

In another embodiment, the NDGA Compounds for inhalation delivery may bedissolved in saline or ethanol before nebulization and administered, asdescribed in Choi, W. S. et al. (2001).

In a further embodiment, the agents herein are also effective whendelivered as a dry powder, prepared in the manner conventional in theart, as described in, for example, Patton, J. S. et al., InhaledInsulin, Adv. Drug Deliv. Rev., 35: 235-247 (1999).

The present invention includes delivery of the NDGA Compounds with theaid of microprocessors embedded into drug delivery devices, such as, forexample, SmartMist™ and AERx™, as described in, for example, Gonda, I.,et al. (1998).

After reading the present disclosure, those skilled in the art willrecognize other disease states and/or symptoms which might be treatedand/or mitigated by the administration of formulations of the presentinvention.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. Examples in the present tenseare prophetic examples.

Example 1 Preparation of a Preparative Batch of Tetra-O-Methyl-NDGA

Tetra-O-Methyl-NDGA, referenced herein as M₄N, was synthesized by thereaction of NDGA with excess dimethyl sulfate in the presence of base,such as potassium hydroxide. The product was isolated by the addition ofwater causing precipitation of the product. The reaction product waspassed through a plug of basic alumina to remove traces of phenolicimpurities, primarily various species of di-O-methyl andtri-O-methyl-substituted NDGA. After the solution of the reactionmixture had passed through the alumina plug, the solvent was removed ona rotary evaporator giving a solid product. This was triturated with2-propanol, filtered and dried in a vacuum oven to give crudetetra-O-methyl-NDGA. Crystallization from 2-propanol gavetetra-β-methyl-NDGA with a purity of greater than or equal to 99.66%.

Step 1: Synthesis of Crude Preparation of Tetra-O-Methyl-NDGA

A 22 L flask fitted with a mechanical stirrer, condenser and inlet forinert atmosphere was set up in a tub for use as a cooling bath. Theflask was placed under an argon atmosphere, and was charged with 484.3grams of NDGA (Western Engineering & Research Co, El Paso, Tex.), and4850 mL of methanol and stirred. To the stirred slurry was added asolution of 387.5 grams of potassium hydroxide in 1210 mL of deionizedwater. The flask containing this reaction mixture was cooled using anice bath, and dimethyl sulfate (1210 mL) was slowly added (dropwise).The addition was controlled to avoid an exotherm. At the end of theaddition, the temperature was about 13° C. The pH of the reaction wasmonitored, and a 50% KOH solution was added in portions during the dayto maintain a basic pH; a total of 1400 mL of 50% KOH solution wasadded. The reaction mixture with excess base gave a pH of about 12, asdetected using pH indicating strips. The solution was dark at basic pH,but became light colored at neutral or acidic pH.

At the end of the day, an additional 600 mL of dimethyl sulfate wasadded, and the reaction mixture was allowed to stir overnight. The nextmorning, the reaction was still basic, and the reaction had progressedto about 90%.

The reaction mixture was quenched by the addition of 4850 mL ofdeionized water, causing the product to precipitate. The product wasisolated by filtration, the filter cake washed thoroughly with water,and the product dried in a vacuum oven at 50° C. for approximately 65 hrto give 539.5 g of the crude product. This product was dissolved in 750mL of methylene chloride, and to this solution was added 375 mL oftoluene. This solution was passed through a short column of 2215 g ofbasic alumina. The alumina was eluted with 12,000 mL of a methylenechloride/toluene solution (2:1). Removal of the solvent in vacuo on arotary evaporator gave a solid residue. This was triturated with 1 L of2-propanol. The resulting slurry was filtered to isolate the solidproduct. This was dried in a vacuum oven at 50° C. under high vacuum forapproximately 21 hr to give 426.7 g (74% yield) of crudetetra-O-methyl-NDGA.

Step 2—Crystallization of Tetra-O-Methyl-NDGA

A 3 L flask with mechanical stirrer, condenser, and inlet was placed ina heating mantle, and was charged with 415.4 g of the product. The flaskwas charged with 1245 mL of 2-propanol, and the stirred mixture washeated to give a mild reflux; a solution was obtained. The heat wasturned off, and the mixture was allowed to cool overnight. Thecrystalline product was isolated by filtration, and the filter cakewashed with 200 mL of cold 2-propanol. The product was dried in a vacuumoven at 50° C. under high vacuum to constant weight giving 404.7 g(70.5% yield overall from NDGA).

Example 2 Preparation of PLGA Nanoparticles Containing NDGA Compounds

The NDGA Compounds can be formulated as a nanoparticle preparation inany manner conventional in the art. For example, the nanoparticles canbe prepared as described in Lamprecht, A. et al. (2001a); and Lamprecht,A. et al. (2001b) and as follows.

The biodegradable polymer poly[DL-lactide-co-glycolide] 50/50 (PLGA)(mol. wt. 5,000 or 20,000) can be purchased from Wako (Osaka, Japan).About 40 mg of a NDGA Compound can be dissolved in 4 ml of methylenechloride containing 250 mg of the polymer poly [DL-lactide-coglycolide]50/50 (mol. wt. 5,000 or 20,000). This solution can thereafter be pouredinto 8 ml of aqueous polyvinyl alcohol solution (1%) and homogenizedwith an ultrasonifier (Ultrasonic Disruptor model UR-200P; Tomy SeikoCo., Ltd., Tokyo, Japan) in an ice bath for 3 min. The methylenechloride can be evaporated under reduced pressure, and the polymerprecipitated. The nanoparticles can be separated from thenon-encapsulated drug and free surfactant by centrifugation (14,000 gfor 5 min). Nanoparticles can be redispersed and centrifuged three timesin distilled water before lyophilization. Before oral administration,the nanoparticles can be re-dispersed in phosphate buffer at pH 6.8.

The nanoparticles can be analyzed for their size distribution and theirsurface potential using a Photal laser particle analyzer LPA 3100(Otsuka Electronics, Osaka, Japan) and a Zetasizer II (MalvernInstruments, Worcestershire, U.K.) respectively. The external morphologyof the nanoparticles can be analyzed with a JEOL JSM-T330A scanningmicroscope (Tokyo, Japan).

Example 3 Preparation of PLGa/Vitamin E TPGS Nanoparticles with NDGACompounds

NPs containing PLGA and another matrix material, d-a-tocopherylpolyethylene glycol 1000 succinate (vitamin E TPGS or TPGS), can be madeas described in Mu, L. and Feng, S. S. (2003), a modified oil-in-watersingle emulsion solvent evaporation/extraction method. In this method,known amounts of mass of polymer and NDGA Compounds are added intomethylene chloride (dichloromethane). The polymer, for example,poly(DL-lactide-co-glycolide (PLGA; L/G=50/50, MW 40,000-75,000;L/G=75/25, MW 90,000-120,000; and L/G=85/15, MW 90,000-120,000), can bepurchased from Sigma (USA). Vitamin E TPGS can be obtained from EastmanChemical, USA. The mixture is stirred to ensure that all the materialsare dissolved. The solution of organic phase is then slowly poured inthe stirred aqueous solution with or without emulsifier and sonicatedsimultaneously at 50 W in pulse mode (Misonix, USA). The formed o/wemulsion can be gently stirred at room temperature (22° C.) by amagnetic stirrer overnight to evaporate the organic solvent. Theresulting sample can be collected by centrifugation, such as at 10,000rpm, 10 min. 16° C. (Eppendorf model 5810R, Eppendorf, Hamburg, Germany)and washed once or twice with deionized water for some samples. Theproduced suspension can be freezed dried (Alpha-2, Martin Christ FreezeDryers, Germany) to obtain a fine powder of nanoparticles, which can beplaced and kept in a vacuum dessicator.

Example 4 Preparation of Liposomes Containing NDGA Compounds

The NDGA Compounds, such as the lipophilic drugs, can be encapsulated inlong acting liposomes by processes conventional in the art. One suchmethod is described in, for example, Sharma, U. S. et al. (1997).

Long-acting liposomes have extended blood circulation time. They aretypically composed of high phase-transition T_(m) lipids, highcholesterol content, and a component such as phosphatidyl inositol,monosialoganglioside (GM₁), or synthetic phospholipids bearing apolyethylene glycol (PEG) headgroup, which provides a steric barrieragainst plasma protein access to the liposome surface.

In an example, liposomes composed of phosphatidylcholine (“PC”):cholesterol (“Chol”): polyethylene glycol conjugated todipalmitoylphosphatidylethanolamine (“PEG-DPPE”) in a molar ratio of9:5:1 can be prepared. The lipids are initially mixed in chloroform, anda thin film of lipid can be produced by evaporation of the solvent. Thelipids are then hydrated in a buffer consisting of NaCl (145 mM),Tris[Hydroxymethyl]-2-aminoethane-sulfonic acid (TES: 10 mM), andethylenediamine tetraacetate (EDTA: 0.1 mM) buffer, pH 7.2. Theliposomes can then be extruded several times through 0.08 μmpolycarbonate filters.

In another example, liposomes composed of distearoylphosphatidylcholine(“DSPC): Chol: PEG-DSPE in at a molar ratio of 9:5:1 can be preparedusing a “remote loading” method as described in Madden, T. D., et al.(1990). This remote loading method allows for encapsulation of highconcentration of NDGA Compounds within the liposome aqueous core.Briefly, a thin film of lipids can be hydrated in ammonium sulfate (250mM, pH 5.5). The lipid suspension can be extruded through 0.08 μmpolycarbonate filters at 60° C. and dialyzed overnight against isotonicsucrose to remove free ammonium sulfate. Hydrophilic NDGA Compounds canbe hydrated in 10% (w/v) sucrose and incubated with the preformedliposomes for 1 hr at 65° C. The preparation can be dialyzed againstisotonic sucrose to remove the minor residual fraction of unencapsulateddrug. This method may yield encapsulation efficiencies of greater thanor equal to 90% of the initial NDGA compounds.

Poly(lactide-co-glycolide)-monomethoxy-poly(polyethylene glycol)(PLGA-mPEG) copolymers of different molar ratios can be prepared by amelt polymerization process under vacuum using stannous octoate ascatalyst, as described in Beletsi, A et al. (1999); and Avgoustakis, K.et al. (2002).

Example 5 Preparation of Intranasal Formulations of NDGA Compounds

The NDGA Compounds can be formulated as a dry powder or an aerosol forintranasal delivery by any methods conventional in the art, such as, forexample, as described in Marttin, E. et al. (1997).

In one embodiment, the NDGA Compound is formulated as a solution withrandomly methylated β-cyclodextrin (“RAMEB”) (degree of substitution1.8) (Wacker, Burghausen, Germany), mannitol or glucose in MQ water,water that is filtered by a Mili-Q UF plus ultrapure water system fromMillipore (Etten-Leur, The Netherlands). This formulation may beadministered as a spray or as drops. The dose of NDGA Compound in theliquid formulation may be from about 1 mg/ml to about 1500 mg/ml, oroptionally from about 10 mg/ml to about 1200 mg/ml, or furtheroptionally from about 100 mg/ml to about 1000 mg/ml, or stilloptionally, from about 200 mg/ml to about 800 mg/ml, or any value thatfalls between these ranges. These liquid formulations can be sprayedinto the nostril or applied as drops.

In another embodiment, the present invention includes lyophilized powderformulations of NDGA Compounds, prepared by dissolving the NDGACompounds and various amounts of RAMEB, lactose, or mannitol in MQwater, and lyophilizing the mixture, such as, for example, overnight.

Example 6 Production of a Biodegradable Polymer Implant

The NDGA Compounds herein can be incorporated into a biodegradablepolymer for implantation into tumors that are not operable. Suchbiodegradable polymer can be made by any method conventional in the art,such as described in Fleming, A. B. and Saltzman, W. M. (2002).Typically, the polymer implant is inserted after removal of the bulk ofthe tumor. One or more wafers of this biodegradable polymer can beimplanted at one time depending on the dose of the compounds desired.The biodegradable matrix of the polymer can be made up of polifeprosan20, a copolymer of 1,3-bis-(p-carboxyphenoxy)propane and sebacic acid[p(CCP:SA)] in a 20:80 molar ratio. To form the polymer for implant,p(CPP:SA) and a compound herein can be co-dissolved in dichloromethaneand spray dried to form spherical particles with a size range of about 1to about 20 μm. The resulting “microspheres” are compression moulded toform wafers of any desired size, such as, for example, about 14 mm indiameter and about 1 mm in thickness. The wafers have a homogeneousstructure consisting of densely packed microspheres surrounded by smallgaps. Concentration of the NDGA Compounds can be in any amountappropriate for the subject to be treated, such as, for example, 3.8%active compound.

Example 7 Preparation of PLGA-mPEG Nanoparticles

PLGA-mPEG nanoparticles containing the NDGA Compounds can be preparedusing the double emulsion method described by Song C. X. et al (1997),with minor modifications. Here, an aqueous solution of the NDGACompounds can be emulsified in dichloromethane in which the copolymer isdissolved, using probe sonication (Bioblock Scientific, model 75038).This water/oil emulsion can be transferred to an aqueous solution ofsodium cholate and the mixture can be probe sonicated. The resultingwater/oil/water emulsion formed can be gently stirred at roomtemperature until evaporation of the organic phase is complete. Thenanoparticles made in this way can be purified by centrifugation andreconstituted with deionized and distilled water. The nanoparticles canthen be filtered such as through a 1.2-μm filter (Millex A P,Millipore).

Example 8 Preparation of Pluronic Micelles Containing NDGA Compounds

Pluronic is a triblock PEO-PPO-PEO copolymer, with PEO representingpoly(ethylene oxide), and PPO representing polypropylene oxide). Thehydrophobic central PPO blocks form micelle cores, while the flankingPEO blocks form the shell or corona, which protects the micelles fromrecognition by the reticuloendothelial system (“RES”). Pluroniccopolymers are commercially available from BASF Corp, and ICI. The NDGACompounds can be introduced into the Pluronic micelles by any methodconventional in the art, as described in, for example, Rapoport, N. Y.,et al. (1999).

Briefly, the NDGA Compounds can be dissolved in PBS or RPMI medium,followed by a short, such as 15 sec, sonication in a sonication bathoperating at 67 kHz. The solution can be kept for about 2 hr at 37° C.,upon which the non-solubilized drug can be removed by dialysis through a1000 D cutoff membrane at 37° C. for about 12 hr against PBS or RPMImedium (dialysis to be done only for 10 and 20 wt % Pluronic solutions).

Example 9 Administration of NDGA Compounds by Implantation

Implantation of the NDGA Compounds herein can be done in any mannerconventional in the art. In one embodiment, implantation is performed asdescribed in Brem, H., and Gabikian, P. (2001). It is preferable thatprior to the insertion of the polymer implant that the tumor besurgically debulked. Further the dura should be closed in a water-tightfashion to eliminate cerebrospinal fluid leakage and to decrease risk ofinfection. It is also desirable to use preoperative anti-convulsants andhigh dose steroids as necessary for neurologic compromise. It is furtherdesirable to continue steroid therapy for at least 2 weekspost-operatively.

Example 10 Delivery of NDGA Compounds in ethanol Via Inhalation

The NDGA Compounds herein can be delivered via inhalation using anyformulation conventional in the art, including as dry powders or asaqueous solutions. The former has the advantage of stability, lowsusceptibility to microbial growth and high mass per puff. Aqueoussolutions offer better reproducibility and avoid the issue of clumping.

In one embodiment, certain of the NDGA Compounds are delivered accordingto the method as described in Choi, W. S. et al. (2001). Depending onthe particular compound and the solubility thereof, the compounds can beformulated to an appropriate concentration in ethanol, such as, forexample in a range of from about 1 mg/ml to about 1000 mg/ml, or anyintervening values in-between, such as, for example, between about 2mg/ml and about 800 mg/ml, or between about 4 mg/ml and about 100 mg/ml,or between about 5 mg/ml and about 50 mg/ml. Aerosol particles of 1-3 μmsize can be generated for maximal deep lung delivery. For bettersolubility of the compounds in ethanol, the compounds herein can befirst lyophilized, then acidified if necessary or desirable, such aswith H₃PO₄. The pH of the resulting composition can be adjusted withNaOH, if desired, such as to pH 7.4. The resulting composition can thenbe lyophilized, suspended in ethanol, sonicated and stirred to produceappropriate submicron size particles. The aerosolized compounds can thenbe administered using a standard commercial nebulizer, such as acompressor (air jet) or an ultrasonic type, or a metered dose inhaler.An example is a PARI LC Jet+ nebulizer (PARI Respiratory Equipment,Monterey, Calif.) in conjunction with a PARI PRONEB compressor. A volumeof about 9 ml can be charged in the reservoir of the nebulizer andnebulized for up to about 10 min.

In another embodiment, the formulation for inhalation can be prepared asdescribed in Wang, D. L., et al. (2000). For example, powdered NDGACompounds can be dissolved in 10:90 (v/v) polyethylene glycol 300:100%ethanol containing 0.5% (w/v) ascorbic acid and 0.5% (w/v)phosphatidylcholine. The drug formulation can then be aerosolized usinga Pari LC-plus nebulizer (Pari, Richmond, Va.) and a subject to betreated can be exposed to the aerosol generated for varying lengths oftime, depending on the dose of the formulation and the desiredconcentration to be achieved. Such periods of time can be about 5minutes, 10 minutes, 15 minutes or longer.

Example 11 Delivery of NDGA Compounds Using Specially Designed Inhalator

The NDGA Compounds can also be formulated in a number of otherpharmaceutically acceptable carriers for inhalation purposes. In thisexample, certain of the compounds herein can be delivered according tothe method of Enk, A. H. et al. (2000). Such compounds can be dissolvedin a solution containing about 5% glucose and 2% human albuminInhalation can then be performed using a specially designed inhalator.(Jetair, Fa. Hoyer, Germany).

Example 12 Delivery of NDGA Derivatives as an Oral Rinse for Treatmentof Oral Lesions

The delivery of NDGA derivatives to the oral cavity involves the use ofan oral rinse using excipients that are conventional in the art, suchas, for example, that described in Armstrong W. B., et al. (2000). TheNDGA derivatives are dispensed as a powder that is reconstituted in anappropriate delivery fluid, such as Roxane Saliva Substitute (RoxaneLaboratories, Columbus, Ohio), immediately before use. Patients thenhold the NDGA derivative suspension in the mouth for about 1 minutebefore expectorating or swallowing the drug mixture. This procedure iscarried out at least once daily for local delivery of NDGA derivativesto the oral cavity.

Alternatively, the delivery of NDGA derivatives to the oral cavity caninvolve an oral rinse formulation such as described in Epstein, J. B.,et al. (2001). Briefly, the NDGA derivatives are prepared in an oralrinse containing about 0.1% alcohol and sorbitol. Patients are providedwith a suitable volume, such as about 5 ml of the rinse, to be rinsed inthe mouth for about 1 minute and expectorated. This procedure is carriedout at least once daily for local delivery of NDGA derivatives to theoral cavity.

Example 13 Arrest of Tumor Growth in Mice After Systemic or OralAdministration of M₄N

In this example, the inventors considerably expanded the cancertherapeutic potential of M₄N by investigating both its anti-tumorefficacy in vivo against several human cancer xenograft models, and itsability to be administered systemically through various routes ofadministration at pharmacologically relevant levels. This example showsthe following: (1) when administered in vivo through differing routes ofsystemic administration including intraperitoneal (IP) injection,intravenous (IV) injection, and oral feeding, M₄N distributesconsistently to various organs and to tumors with little or no apparenttoxicity to mice; (2) systemic IP administration of M₄N effectivelyretards the in vivo growth of xenografts from 4 human cancer cell types:MCF-7 breast adenocarcinoma, Hep3B hepatocellular carcinoma, HT-29colorectal carcinoma, and LNCaP prostate carcinoma; and systemic oraladministration of M₄N effectively suppresses the growth of LNCaPxenograft tumors—only LNCaP tumors have thus far been assessed in oraladministration efficacy studies.

Cell Lines and Culture Conditions. Human tumor cell lines were purchasedfrom ATCC (Mannassas, Va.). The human hepatocellular carcinoma cellline, Hep 3B, and the human breast epithelial adenocarcinoma cell line,MCF-7, were grown in Eagle's Minimal Essential Media +10%FBS+penicillin+streptomycin. The human colorectal adenocarcinoma cellline, HT-29, was grown in McCoy's 5a Medium+10%FBS+penicillin+streptomycin. The human prostate carcinoma cell line,LNCaP was grown in RPMI 1640+10% FBS+penicillin+streptomycin.

Mice. Female ICR mice, 6-8 weeks of age, were purchased from HarlanSprague Dawley (Indianapolis, Ind.). C57bl/6 mice were purchased fromCharles River Laboratories (Wilmington, Mass.). Athymic (thy⁻/thy⁻) nudemice, males and females 5-6 weeks of age, were purchased from CharlesRiver Laboratories and were housed in a pathogen-free room undercontrolled temperature and humidity in accordance with InstitutionalAnimal Care and Use Guidelines. C57bl/6 mice bearing C3 cell-inducedtumors were prepared as described in Kim, E. H. et al. (2004).

Xenograft Assay of Human Tumors. Athymic nude mice were implantedsubcutaneously in their flanks with 2.5×10⁶ Hep3B cells, 2×10⁶ LNCaPcells, 1×10⁷ HT-29 cells, or 2×10⁶ MCF-7 cells. After the tumorsexhibited a mean diameter of 7-8 mm, the mice were assigned to one oftwo groups: a control group receiving vehicle only, and a groupreceiving M₄N dissolved in the Cremaphor EL-ethanol-based solventsystem. Assignment was made so that both the control group and theexperimental group contained mice bearing tumors of comparable sizes.

M₄N was dissolved in 6% Cremaphor EL, 6% ethanol, 88% saline asdescribed in Loganzo et al. (2003). Mice received a single daily 100 μLi.p. injection containing 2 mg of M₄N for 3 weeks. The control micereceived an equal volume of the vehicle. Tumors were measured in twoperpendicular dimensions (L and W) once every seven days, and the tumorvolumes were calculated according to the following formula:V=(L×W/2)³×π/6. The results from the individual mice were plotted asaverage tumor volume versus time. Statistical significance of the meandifferences in tumor volume was assessed by Student's t-test. At thetermination of the experiment, tumor biopsies were collected forimmunohistological analysis of cdc2 and survivin expression.

M₄N Tissue Distribution Studies Using ³H-M₄N. Harlan ICR mice or C3cell-induced tumor-bearing C57bl/6 mice were injected via tail vein orintraperitoneally with 100 μL of Cremaphor-ethanol based solventcontaining 100 μCi of tritiated M₄N and 60 mM of cold M₄N. At thespecified time post-injection, the mice were sacrificed, the organs andblood were collected, weighed, then dissolved overnight in 4 M guanidineisothiocyanate (GITC). The insoluble pellet was then further extractedwith EtOH. Tritium content of both the GITC extract and the EtOH extractwas measured on a Packard scintillation counter and the quantity of M₄Nin each organ was calculated based on the specific activity of theinoculum.

Tissue Distribution and Toxicity Analysis Following Short-Term andLong-Term Oral Feeding. For short-term feeding experiments, 30 mg of M₄Ndissolved in 300 μL castor oil was orally administered to each of 6mice. At 2 h, 4 h, and 8 h post-administration time points, 2 mice weresacrificed, the organs and blood were collected, and the M₄N extractedand quantitated as described below. In long-term feeding experiments,mice were fed food balls consisting of M₄N dissolved in corn oil andBasal Mix (Harlan Teklad; Madison, Wis.; Cat. # TD 02273) for 14 weeks.Food balls weighed 9 g and contained 242 mg M₄N each. Two mice, one maleand one female, were reserved for long-term drug retention studies; andfourteen mice, both male and female, were used for long-term drugtoxicity studies. At the end of feeding, mice were sacrificed, theorgans and blood were collected, and the M₄N extracted and quantitatedas described below.

M₄N Extraction and HPLC Analysis Following Oral Feeding. Organs andblood were harvested from M₄N-fed mice, then frozen overnight at −80° C.Prior to freezing, gastro-intestinal organs (stomach, small intestine,colon) were cut open longitudinally and washed thoroughly with PBS toremove any contents. The following day, organs were cut into smallpieces on dry ice with a razor blade, dried in a Speed-vac, then crushedinto a rough powder using a mortar and pestle. Samples were extractedovernight in 100% ethanol with shaking Samples were centrifuged and thesupernatant collected. Pellets were extracted two more times in 100%ethanol overnight with shaking. The pooled ethanol extracts wereevaporated on bench top for several days, then re-extracted with ethylacetate, and dried completely in a Speed-vac. The dried samples werethen analyzed quantitatively by HPLC and M₄N was identified by massspectroscopy using pure M₄N as a standard.

In samples from short-term fed mice, dialysis was performed to furtherpurify M₄N from the tissue extracts. Dried ethanol extracts wereredissolved in 1.5 mL 100% EtOH and centrifuged for 5 min. Thesupernatant was collected and the pellet was resuspended in 0.4 mLethanol and centrifuged again. The supernatant was pooled with theprevious supernatant, then dialyzed overnight against 150 mL 100% EtOH.The dialysates were dried on bench top and in a speed-vac, then analyzedby HPLC.

HPLC Quantitation of M₄N. Samples from a single mouse at each time pointwere sent to KP Pharmaceuticals (Bloomington, Ind.) for HPLC analysis.HPLC conditions were described as follows: 35%:0.1% TFA in H₂O,65%=“CAN.” The M₄N standard was prepared by diluting 10.01 mg M₄N in 100mL of CAN, then sonicating for 5 min. (2002 ng/injection). The sampleswere prepared by adding 400 μL EtOH and sonicating for 2 min. or untilcomplete dissolution was achieved. The injection volume for the sampleswas 100 μL.

M₄N is Distributed Systemically to Various Tissues and With NoDetectable Toxicity Following Intraperitoneal, Intravenous, and OralAdministration

Systemic Distribution of M₄N Following a Single Intraperitoneal orIntravenous Administration

Previous studies demonstrated substantial tumoricidal activity followinglocalized intratumoral injection of M₄N into C3 cell-induced tumors inmice, as described in U.S. Pat. No. 6,608,108. Yet, with few exceptions,the clinical use of nonsystemic intratumoral chemotherapy is rare evenfor high mortality cancers characterized by well defined primary lesionsi.e. breast, colorectal, lung, and prostate. Rather, the conventionalwisdom and standard of care in clinical oncology remains surgeryfollowed by systemic chemotherapy and/or radiation as deemed appropriateto the clinical situation. Because the effective treatment of manyprimary tumors as well as metastatic disease requires systemic delivery,the ability to distribute M₄N systemically in vivo was assessed.

A mixture of tritiated and cold M₄N was dissolved in a 6% Cremaphor EL,6% ethanol, 88% saline solvent then injected intraperitoneally (i.p.)and intravenously (i.v.) via tail vein into mice. At 3 hourspost-injection, the organs and blood were harvested and weighed, and theM₄N was extracted. The tritium content of the extracts from each organwas measured on a Packard scintillation counter and the quantity of M₄Nin each organ was calculated based on the specific activity of theinoculum. As shown in FIGS. 1A and 1B, M₄N was successfully distributedto various organs at 3 hours post-injection by both i.p. and i.v. routesof administration. Interestingly, very similar profiles of tissuedistribution were obtained despite the different routes ofadministration, thus indicating a non-random, perhaps regulatedmechanism of drug dispersal. Corroborating this, a very similar profileof distribution was observed using an oral route of administrationdescribed below. The majority of the recovered radioactivity localizedto the gastrointestinal tract organs (FIG. 1A): the stomach, smallintestine, caecum, and large intestine, in the range of 3 μg to 20 μg ofM₄N per gram of tissue. Significant quantities of M₄N were also presentin the liver and fat, and lower concentrations in the range of 150 to400 ng per gram tissue (FIG. 1B) were detected in the brain, kidneys andspleen. Little or no M₄N, however, was detected in the heart or theblood at 3 hours post-injection. In conclusion, M₄N may be safely andsystemically administered to various specific tissues via i.p. or i.v.injection.

The previous experiment demonstrated that M₄N may be deliveredsystemically and relatively rapidly to various tissues at a single timepoint. To determine the distribution of M₄N in tissues over timefollowing a single application, and also to assess the ability todeliver M₄N to a distant tumor, six C3 cell-induced tumor bearing mice(A-F) were treated i.p. with ³H— M₄N as described above. At 4 hours, 6hours, 18 hours, and 6 days post-injection, the quantities of ³H— M₄N invarious tissues and the tumors were measured. The results shown in FIG.2 confirmed the ability to distribute M₄N systemically, with themajority of M₄N again localizing to the GI tract organs, fat and liver,and lesser amounts detected in the brain and kidneys. Interestingly, andnot apparent in the previous 3 hour injection, the fat and spleenexhibit a rapid increase in M₄N levels between 4 hours and 6 hours. Asignificant, although relatively low amount of M₄N, 294 ng M₄N per gramof wet tumor, was measured in the tumor at 6 hours post-injection. Thechanges in tissue distribution of M₄N following initial application showan increase in M₄N levels in these tissues from 0 to 6 hours with a peakoccurring at approximately 6 hours. At 18 hours, M₄N levels hadsubstantially decreased, and at 6 days post-injection, althoughsignificant M₄N levels could still be detected in most tissues, M₄Nlevels had decreased to 5-10% of levels seen at 6 hours.

Systemic Tissue Distribution Following Short-Term and Long-Term OralFeeding and In Vivo Toxicity Evaluations.

The previous experiments demonstrated that M₄N can be systemicallydistributed in vivo by i.p. and i.v. injection with no apparenttoxicity. The convenience and ease of oral administration, however,especially in the case of long-term post-surgical adjuvant treatment,would considerably facilitate drug administration to patients and wouldimprove patient quality of life. Thus, in addition to i.p. and i.v.administration, the ability to systemically distribute M₄N by oraladministration was also investigated. In both short-term (<8 hours)feeding experiments and long-term (14 weeks) feeding experiments, M₄Nlevels in various tissues and their in vivo toxicity was assessed. Inshort-term experiments, mice were fed 30 mg of M₄N dissolved in castoroil (100 mg M₄N/mL castor oil), and at 2, 4, and 8 hours post-feeding,the quantity of M₄N present in various tissues was determined by HPLC.As shown in Table 1, a relatively very low quantity of M₄N (<2 ng pergram tissue) was found in each tissue at 2 hours post-feeding. Between 2and 4 hours post-feeding, most organs including the liver, pancreas,kidneys, seminal vesicles, small intestine, stomach, large intestine,caecum, and blood exhibited a large increase in M₄N levels. At 4 hours,as was seen in the i.p. and i.v. administrations, most of the M₄Nlocalized to the gastro-intestinal tract organs, in the range of 4 ng to45 ng of M₄N per gram of tissue. Significant quantities of M₄N were alsopresent in the pancreas, and lower concentrations in the range of 0.1 ngto 2 ng per gram tissue were detected in the heart, liver, seminalvesicles, blood, and bladder. At 8 hours post-feeding, M₄N levels haddecreased in nearly all organs, and most of the organs had been clearedof M₄N. In conclusion, M₄N was distributed transiently to various organsfollowing a single oral administration of 30 mg of M₄N. M₄N levelspeaked at roughly 4 hours post-feeding, and M₄N concentrations weresignificantly lower than seen in i.p. and i.v. single administrations.

TABLE 1 M₄N (ng)/organ dry weight (g) M₄N (μg/g) Organ 2 hours 4 hours 8hours 14 weeks Heart 0.83 0.11 1.16 12.62 Liver 0.05 0.52 0 5.89 Lungs0.43 0 2.7 23.09 Pancreas 0.57 24.69 2.97 28.81 Kidneys 0.12 0.61 0 8.33Seminal Vesicles 0.21 0.37 0 16.79 Small Intestine 0.09 8.42 5.53 906.27Stomach 0.23 45.03 33.9 409.27 Lg. Intestine 0.24 8.24 2.68 350.88Caecum 1.11 4.38 6.55 440.23 Spleen 1.1 0.16 0 302.78 Blood 0.5 2.08 023.13 Bladder 2.34 1.69 0 6.24 Fat 0.11 0 0 17.41

The objective of the long-term feeding experiments was to measure thesteady state levels of M₄N in various mouse organs following continuousoral administration for 14 weeks. Food balls weighing approximately 9 gand containing approximately 280 mg M₄N were continually fed towild-type mice for 14 weeks. A single 9 g food ball is consumed by asingle mouse in about 3 days, which translates to 93.3 mg of M₄Nconsumed or administered daily. HPLC quantitation showed that oraladministration had systemically distributed M₄N to all organs analyzed;and surprisingly had accumulated in all organs to concentrations greatlyexceeding those seen previously for i.p., i.v., and oral one timeadministrations. Between 350 μg and 900 μg M₄N per gram tissue wasmeasured in the GI tract organs and the spleen; 15 μg/g to 30 μg/g M₄Nwas measured in the lungs, pancreas, seminal vesicles, blood, and fat;and 5 μg/g to 13 μg/g M₄N was measured in the heart, liver, kidneys, andbladder.

Despite the high levels of M₄N present in various organs followingsystemic long-term oral administration, no signs of toxicity were seenas determined by daily evaluation of activity and overall body weightchange (FIG. 3).

Systemic M₄N treatment inhibits the in vivo growth of human tumorxenografts.

Based on (1) our cell culture analyses showing that M₄N will effectivelyprevent the growth of various human tumor cells, and (2) our in vivoobservations that M₄N can be distributed systemically at non-toxicdoses, we investigated whether systemic administration of M₄N wouldinhibit the in vivo growth of human tumors. Athymic nude mice wereimplanted s.c. in each flank with MCF-7 breast adenocarcinoma cells,Hep3B hepatocellular carcinoma cells, HT-29 colorectal carcinoma cells,and LNCaP prostate carcinoma cells. Most mice developed tumors in bothflanks, although some developed a single tumor. When tumors attained amean diameter of 7-8 mm, mice received for three weeks a single dailyi.p. injection containing 2 mg of M₄N dissolved in 100 uLCremaphor-ethanol based solvent. Control mice received vehicle only.Tumors were measured in two perpendicular dimensions (L and W) onceevery seven days, and tumor volumes were calculated according to theformula: V=(L×W/2)³×π/6.

As shown in FIG. 4, systemic M₄N treatment for 21 days resulted instatistically significant (p<0.05) reductions in mean tumor growth inall four tumor types. After 21 days of systemic M₄N treatment, MCF-7tumors were reduced 74% in mean tumor volume to being only 25.5% of themean volume of the control tumors (Table 2); HT-29 tumors were reduced70% in mean volume; Hep 3B liver tumors were reduced 80%; and LNCaPprostate tumors were reduced 53%.

TABLE 2 Tumor Volume Increase (%) - 21 Days Treatment Tumor M₄N- Ratioof Mean M₄N-Treated Tumor Size Type Treatment Control to Control TumorSize McF-7 84% 624% 25.5% Ht-29 787% 2920% 29.4% Hep 3B 113% 1001% 19.3%LNCaP 25% 171% 46.3%

Table 3 shows the total number of tumors in each treatment group, andcategorizes each based on whether there was an overall increase ordecrease in size over the 21 days of treatment. In all four tumor types,100% of the control tumors each exhibited an increase in tumor size.However, among the M₄N-treated mice, 7 out of 10 MCF-7 tumors decreasedin size; 2 out of 7 Hep3B tumors decreased in size, 3 out of 11 HT-29tumors decreased in size, and 9 out of 11 LNCaP tumors decreased in sizefollowing 21 days of systemic M₄N treatment. In sum, 100% of the 47control tumors increased in size, whereas 53%, or 21 out of 39,M₄N-treated tumors decreased in size following 21 days of systemictreatment; of the remaining 18 out of 39 M₄N-treated tumors, althoughincreasing from their starting tumor size, most exhibited interruptedgrowth during the 21 days of treatment. Despite the significanttumoricidal effect observed for each tumor type, body weights and thegeneral health of the mice were monitored over the 21 day course oftreatment and indicated no toxicity in any of the mice.

TABLE 3 Treatment Total # Tumors IOTV DOTV Tumor Type Control MCF-7  7 70 Hep 3B  6 6 0 HT-29 24 24  0 LNCaP 10 10  0 47 (100%) 47 (100%) 0 (0%)Tumor type M₄N MCF-7 10 3 7 Hep 3B  7 5 2 HT-29 11 8 3 LNCaP 11 2 9 39(100%) 18 (46%)  21 (53%) IOTV: Increase in Volume from the OriginalTumor Volume DOTV: Decrease in Volume from the Original Tumor Volume

Example 14 Safety Studies in Humans

In this example, the inventors demonstrated clear safety and efficacy ofNDGA derivative delivery in humans as a therapy for head and neckcancer. This example describes the results of two separate clinicalstudies that spanned range of patient ages, stages of diseasedevelopment, and two different treatment methods. This exampledemonstrates the following: (1) M₄N can be delivered by escalating dosesup to about 495 mg weekly for three weeks or at dosages of 20 mg per dayfor up to five days without drug-related toxicity. This daily deliveryof M₄N can be with or without concomitant therapy with G₄N at a dose of20 mg per day for up to five days and is followed by surgical resectionof the lesion; (2) Both of these treatment methods delivered over 80%efficacy in terms of induction of necrosis in patients that completedthe treatments; (3) In long-term follow ups of the ex-US study 64% ofpatients remained disease free and also free from long term effects ofNDGA-derivative exposure.

US Phase I Intratumoral Head and Neck Cancer Study:

A Phase I clinical study has been completed under a US IND. Mean subjectage was 66 years (range 53 to 82 years). Eight male subjects and onefemale subject participated. Mean weight was 139 lbs. (range 102 to 219lbs.). All patients were diagnosed with refractory head and neckcarcinomas.

Nine (9) subjects were dosed with an intratumoral dose of M₄N givenweekly for three weeks, at doses of 5 mg/cm³ tumor volume (2 subjects),10 mg/cm³ (2 subjects) 15 mg/cm³ (3 subjects) and 20 mg/cm³ (2subjects). Doses up to 495 mg weekly for three weeks were administered.

Three subjects completed the study per protocol. Two subjects died onstudy from causes considered unlikely to be related to study medication.One subject withdrew consent after receiving three doses of M₄N as hecould not travel to meet protocol requirements. One withdrew consentafter experienced severe radiating pain on injection associated with anaccidental perineural dose. One was withdrawn after a single dose as histumor was considered to be too close to the carotid artery to allow asafe second dose. One was withdrawn as a result of tumor progression.Dosing related adverse events were otherwise minor and included mild ormoderate pain on injection (4 subjects). No other adverse events wereattributed to M₄N administration. Sporadic, and non-reproducible mildelevations in LFTs were seen in 2 subjects, which resolved while stillon therapy. No changes attributable to drug were seen in hematologyparameters.

Six (6) Serious Adverse Events (SAE) were reported in four (4) subjects.Serious adverse events included supraventricular tachycardia (twoepisodes on separate occasions in one subject), pneumonia, dehydrationand death from tumor progression (one subject), and death 19 days afterstudy (cause unknown). In all cases, the SAE's were considered unlikelyor not related to study medication.

In 5 of 6 subjects receiving three doses, drug related tumor necrosisoccurred after injection. No damage occurred to healthy tissuesurrounding the tumor. Fistula formation developed where tumors werefull thickness. Tumors also were noted to have softened, or “pancaked”,but residual tumor at the margins continued to grow, suggesting thatsystemic administration may be more appropriate. Tumor volume reductionwas radiologically confirmed in three of the six patients completingthree doses. Dosing was generally well tolerated.

Example 15 Safety Studies in Beagle Dogs Following 14-Day IntravenousInfusion of M₄N

In this example the Maximal Tolerable Dose (MTD) of two differentformulations of M₄N [Cremaphor-Ethanol (CET) or Dimethyl Sulfoxide(DMSO)] to male and female beagle dogs was determined. This exampleshows that M₄N was safely administered by intravenous infusion into dogsover four hours at doses up to 10 mg/kg with a CET vehicle or up to 100mg/kg with a DMSO vehicle. Blood levels of up to 14,000 ng/ml M₄N wereachieved with these formulations with minimal toxicity.

Vascular Access Port (VAP) Implantation Surgery for M₄N-CET Group

VAPs were implanted into beagle dogs such that the tip of the infusioncatheter was situated at the level of the superior vena cava. Dogs weretreated prophylactically with an analgesic and antibiotic on the day ofsurgery and with antibiotics and/or analgesics following surgery(according to Gene Logic Inc. SOP Nos. 324.0.2, 325.0.1, and 326.0.2, asappropriate.) Other treatments were provided as recommended by the staffveterinarian. The catheter lines were flushed with saline during thepostoperative recovery period with a frequency deemed appropriate by theStudy Director.

Although VAPs were implanted into dogs that were assigned to receiveinfusion of M₄N-DMSO, however, DMSO was found to be not compatible withthe infusion catheter attached to the VAP inside the animals. Thus theM₄N-DMSO group animals were administered with M₄N-DMSO with eightintravenous injections via the non-VAP jugular vein every 30 minutesover a 4-hour period. This frequency of delivery mimicked the deliveryof the test article using the infusion pump.

TABLE 4 Group Designation and Dose Levels Number Dose Infusion Injectionof Level Rate Volume Treatment Dogs (mg/kg) (mL/kg/hr) (mL) DurationM₄N-CET 1M, 1F 0 M (1.7), 2 hours F (1.3) 1 M (1.7), 2 hours F (1.4) 5 M(0.7), 4 hours F (0.6) 10 M (1.8), 4 hours F (1.3) M₄N-DMSO^(a) 1M, 1F 0M (0.17), 4 hours F (0.12) 10 M (0.16), 4 hours F (0.13) 50 M (0.83), 4hours F (0.66) 100 M (1.78), 4 hours F (1.35) M₄N-DMSO^(b) 1M, 1F 200 M(4.1), ~1 hour F (2.8) ^(a)8 intravenous injections every 30 minutesover 4 hours ^(b)Additional animals to determine potential toxicity, Mreceived 3 injections, F received 2 injections

Animals from the CET group were observed during the entire infusionperiod and for at least one hour following end of infusion. The dogs inthe DMSO group were observed throughout the jugular vein injectionperiod and for at least one hour following the last (eighth) injection.

Blood Sample Collection for Toxicokinetic (TK) Analysis

Blood samples from the CET group animals were collected via the jugularvein on Study Day (SD)1, SD 3, SD 6, and SD 8 at the followingtimepoints: predose, 0.25, 0.5, 1, 2, 4, 8, and 16 hours following thecompletion of the approximate 4-hour infusion.

Blood samples from the DMSO group animals were collected via thecephalic vein on SD 1, SD 3, SD 6, and SD 8 at the following timepoints:predose, 0.25, 0.5, 1, 2, 4, 8, and 16 hours following the finalinjection dose of M₄N-DMSO.

Blood samples collected from both groups of animals were processed forplasma and serum for TK analysis.

TK Analysis

The plasma and serum samples were sent to MedTox Laboratories, theSponsor's designated laboratory for TK analysis. TK analysis of M₄Nplasma and serum concentration-time data was performed using a validatedmethod (M200406) by MedTox Laboratories and analyzed by noncompartmentalmethods to obtain estimates of toxicokinetic parameters (where dataallow), but not necessarily limited to, Cmax, Tmax and AUC.

Study Day 1 (SDI):

a) M₄N-CET Group

Male dog: reacted to CET infusion with erythema, hives, itchiness,emesis, diarrhea, and general lethargy in the first hour and a half. Hebegan to recover after that, started walking around, drinking water. Hebehaved normally soon following end of infusion. Female dog: reactedsimilarly to the male dog except without emesis and diarrhea. Herallergic reactions were also less severe than the male. She behavednormally soon following end of infusion.

b) M₄N-DMSO Group

Male dog: reacted to DMSO with slight erythema, slight itchiness,otherwise normal. Behavior was normal soon following end of lastinjection. Female dog: reacted similarly to the male dog. Her behaviorwas normal soon following end of last injection.

All 4 dogs survived the infusion of their respective vehicle treatment.They all appeared fine and behaved normally following treatment.

Study Day 3 (SD3):

a) M₄N-CET Group

Male dog: reacted to M₄N-CET (1 mg/kg) infusion with slight erythema,hives, itchiness. The reactions this day were milder than those on SD1.In particular, the animal did not have emesis, diarrhea, or lethargy, hewas more alert than on SD1. He behaved normally soon following end ofinfusion. Female dog: her reactions to the M₄N-CET (1 mg/kg) infusiontoday was even milder than those observed on SD1. Her allergic reactionsincluded mild erythema and itchiness, but she was generally quite alertthroughout the 4-hr infusion period. She behaved normally soon followingend of infusion.

b) M₄N-DMSO Group

Male dog: There was no adverse clinical reaction exhibited by this dog.There was, as expected, some irritation at the injection sites along thejugular vein.

Female dog: There was no adverse clinical reaction exhibited by thisdog. There was, as expected, some irritation at the injection sitesalong the jugular vein.

All 4 dogs survived following administration of their respective testarticle treatment. They all appeared fine and behaved normally followingtreatment.

Study Day 6 (SD6):

a) M₄N-CET Group

Male dog: Similar to the previous two dosing days, this animal reactedto the infusion with slight erythema, hives, and itchiness. Theintensity of the reactions was certainly no more than the reactions onSD3. He did not vomit or had diarrhea, was generally alert throughoutthe infusion period. He behaved normally soon following end of infusion.

Female dog: Consistent with her reactions to previous dosings, shetolerated today's infusion better than the male dog, she still had milderythema and itchiness, but she was quite alert. She behaved normallysoon following end of infusion.

b) M₄N-DMSO Group

Male dog: This dog was successfully injected intravenously with M₄N-DMSOvia the non-VAP jugular vein for the first 3 dosing intervals (½ hrbetween doses). As with the previous dosing days, this dog did not showany adverse clinical signs or symptoms following each injection. Priorto the fourth injection, the technicians noticed a swelling “the size ofan egg” around the injection site. Subcutaneous misdose could be ruledout because it would have been easily detected during the 3rd injection.It was most likely a hematoma as a result of slow extravasation of bloodthrough the injection site. This animal did not receive any more dosingfollowing the third injection, however, blood samples were collected,the exact time points of the blood collection post-third injection dosewas clearly documented. The hematoma resolved within two hours andgentle massaging of the injection site area did not irritate the animal.

Female dog: This dog was successfully injected with M₄N-DMSO for theentire 8 repeated injections over 4 hours. Similar to the previousdosing days, this dog did not show any adverse clinical signs orsymptoms

Study Day 8 (SD8):

a) M₄N-CET Group

Both male and female dogs received full dose. Their reactions to thishigh dose were similar to those exhibited in previous dosing days, whichinclude erythema, hives, and itchiness. No vomiting or diarrhea wasnoted. The animals behaved normally soon following end of infusion.

b) M₄N-DMSO Group

Both the male and female dogs received full dose. Their reactions to thehigh dose were similar to those on previous dosing days. There appearedto be more G. I. irritation as both dogs showed some retching reactionwithout vomiting, they were more lethargic than usual. However, bothdogs survived the full high-dose administration regimen and appeared tohave recovered following the end of dosing.

Additional dose (200 mg/kg) for M₄N-DMSO Group

Since animals dosed with M₄N-DMSO at 100 mg/Kg did not show adverseclinical signs or symptoms, two spare dogs (1 male, 1 female) were dosedwith M₄N-DMSO at 200 mg/Kg. At 200 mg/Kg, the female dog experienceddifficulty breathing (with nasal frothing) after only the first of eightdoses, she soon collapsed but was able to recover for the second dose.After the second dose, her reaction was similar but even more severe.Thus the staff veterinarian suggested euthanizing the female dog. Themale dog was slightly more tolerant but exhibited similar difficultybreathing signs and collapsing symptoms. He received a total of threedoses and the staff veterinarian suggested further dosing be stopped.

There were no post-dose TK analysis for this additional dosing, allpre-dose blood samples collected today were discarded.

TK Analysis

a) M₄N-CET Group

TABLE 5 M₄N-CET - SERUM RESULTS (NG/ML) Animal No./Sex Dose Predose 15min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr 10828F  1 mg/kg <2 >100 >100 78.5948.32 39.75 9.88 4.33 10827M  1 mg/kg <2 >100 >100 53.78 32.72 26.657.19 4.63 10828F  5 mg/kg <1 >100 >50 >50 >50 34.40 19.45 11.57 10827M 5 mg/kg <1 >50.0 >50 >50 45.35 19.99 12.98 6.71 10828F 10 mg/kg<20.0 >1000 >1000 705.47 285.10 187.82 133.50 52.94 10827M 10 mg/kg63.76 >1000 >1000 783.86 431.21 158.20 117.00 60.36In general, intravenous infusion of M₄N-CET at different dose levels for4 hours resulted in extremely high serum concentrations at the earlytime points and peaked at 30 minutes following end of infusion (Table5). The serum concentrations of the test article reduced over the next15 hours.

b) M₄N-DMSO Group

TABLE 6 M₄N-DMSO - SERUM RESULTS (NG/ML) Animal No./Sex Dose Predose 15min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr 10831F  10 mg/kg <2 594.98 398.95436.92 238.20 97.92 43.80 38.39 10832M  10 mg/kg <2 516.8 533.07 348.7252.86 317.13 78.87 56.27 10831F  50 mg/kg 3.49 1136.51 474.95 673.4 241144 101 58.8 10832M  50 mg/kg 19.91 NR NR NR 234.15 79.11 65.79 45.810831F 100 mg/kg 21.47 8688.10 8163.68 7696.48 2624.2 1021.05 459.82222.22 10832M 100 mg/kg 38.22 10477 14088 7498.88 3878.86 3468.19 814.24593.83

In general, repeated intravenous injection of M₄N-DMSO at different doselevels for 4 hours resulted in extremely high serum concentrations. Theserum concentration data reported in Table 6 are the results followingsystematic dilution of the serum to accommodate detection range. Theresults showed that in general, the serum concentration of M₄N-DMSO washigh in the early time points and peaked at 30 minutes following thelast injection. The serum concentrations of the test article reducedover the next 15 hours. Based on the serum concentrations from thisgroup, the half-life of M₄N-DMSO, when administered by repeatedintravenous injection, was approximately 1.5 to 2 hours. It isnoteworthy that from the pre-dose serum concentrations of M₄N-DMSO overthe course of this MTD phase, there was a slight build-up of the testarticle in the blood, but this retention was generally less than 0.3% ofthe highest serum concentration.

The purpose of the MTD phase of this study was to determine the maximumtolerable dose of two different formulations of M₄N (Cremaphor-Ethanolor DMSO) to male and female beagle dogs. The group of animals thatreceived M₄N-CET reacted with itchiness, erythema, hives, andsleepiness; clinical signs and symptoms consistent with the effects ofCremaphor-Ethanol. Animals that received repeated injections of M₄N-DMSOshowed some irritation at the injection site and minor retching at 100mg/kg. However, both animals collapsed following 2 or 3 injections ofM₄N-DMSO at 200 mg/kg. TK analysis from this group suggested a half lifefor M₄N-DMSO in the range of 1.5-2 hours. There was minor build up ofthe test article over the course of the MTD phase, however, thisretention only amounted to less than 0.3% of the maximum serumconcentration. In conclusion, the MTD phase of this study was a successas the dose level that resulted in significant adverse clinical signsand symptoms was identified, thus the objective of this phase wasachieved.

Treatment of Therapy-Refractory Acute Lymphoblastic Leukemia with NDGADerivatives.

NDGA derivatives can be used as a treatment for acute lymphoblasticleukemia (ALL). Such use can be assessed via a method similar to thatdescribed by Uckun, F. M. et al. (1999). For example, patients with ALLhaving relapses following frontline or salvage chemotherapy or havingfailed induction chemotherapy can be subject of treatment with the NDGAderivative.

In one example, the NDGA derivative, G4N, is formulated as a sterilesolution, for example, 1 mg/ml in 150 mM sodium chloride and 40 mMsodium phosphate (pH 7.4). For intravenous administration, the drug isfurther diluted in 10 ml/kg (up to about 100 ml) normal saline. Eachtreatment consists of a continuous infusion for a period of timedepending on the half-life of the particular NDGA derivative, forexample, a 1-hr infusion, given 15-20 minutes after premedication withstandard doses of diphenhydramine (Benadryl) and acetaminophen(Tylenol). Patients are treated with one or two courses of therapy, eachcomprising either 10 consecutive days of treatment or three weeklycycles of 3 consecutive days each for a total of nine doses. The dosesto be given depend on the characteristics of the patients and the statusof the diseases and can be, for example, one of 0.1 mg/kg/day, or 0.18mg/kg/day, or 0.32 mg/kg/day or higher.

The successfulness of NDGA therapy for the treatment of ALL issummarized by classifying patient cases as complete remission, partialresponse, progressive disease, or stable disease. Complete remission(“CR”) of ALL in response to NDGA derivative treatment is defined as theachievement of M1 bone marrow status (<5% blasts) by day 28 of therapywith a granulocyte count of 1×10³/μl, a platelet count of 100×10³/μl(>50,000/μl without transfusions for patients post-BMT), a hemoglobinlevel of 10 g/dl, and absence of circulating leukemia cells in theperipheral blood or any evidence of extramedullary disease. Partialresponse (“PR”) is defined as a complete disappearance of peripheralblasts and achievement of M2 bone marrow status (5-25% blasts) by day 28of therapy. Progressive disease (“PD”) is defined as an increase of atleast 25% in the absolute number of circulating blasts, or developmentof extramedullary disease. Stable disease (“SD”) is defined as a lack ofchange in status of any of the parameters that would result in CR, PR,or PD. NDGA derivatives are seen to have some inhibitory effect on thedevelopment of PD.

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It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A method for treatment of a disease in a subject comprising: (a)providing a composition comprising at least one catecholic butane otherand a pharmaceutically acceptable carrier or excipient, wherein thewherein the catecholic butane has the formula:

wherein R₁ and R₂ are independently —H, a lower alkyl, a lower acyl, analkylene or an unsubstituted or substituted amino acid residue or saltthereof; R₃, R₄, R₅, R₆, R₁₀, R₁₁, R₁₂ and R₁₃ are independently —H or alower alkyl; and R₇, R₈ and R₉ are independently —H, —OH, a loweralkoxy, a lower acyloxy, or any two adjacent groups together may be analkyene dioxy, or an unsubstituted or substituted amino acid residue orsalt thereof, provided that the catecholic butane is not NDGA. (b)administering the composition to the subject systemically by a route ofadministration selected from the group consisting of: oraladministration; inhalation administration; intra-arterialadministration, with or without occlusion; intracranial administration;intraventricular administration; intravenous administration;intramuscular administration; implantation administration; and centralvenous administration. (c) wherein the disease is selected from thegroup consisting of: acute lymphoblastic leukemia, acute myeloidleukemia, adrenocortical carcinoma, anal cancer, astrocytoma, bile ductcancer, bladder cancer, bone cancer osteosarcoma/malignant fibroushistiocytoma, brain stem glioma, brain tumor ependymoma, brain tumormedulloblastoma, breast cancer, carcinoid tumor gastrointestinal,carcinoma adrenocortical, carcinoma islet cell, cervical cancer, chroniclymphocytic leukemia, chronic myelogenous leukemia, clear cell sarcomaof tendon sheaths, colon cancer, cutaneous T-cell lymphoma, endometrialcancer, epithelial cancer ovarian, esophageal cancer, Ewing's family oftumors, extragonadal germ cell tumor, extrahepatic bile duct cancer, eyecancer, intraocular melanoma, eye cancer retinoblastoma, gallbladdercancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germcell tumor extragonadal, germ cell tumor, ovarian tumor, gestationaltrophoblastic tumor, glioma, hairy cell leukemia, hepatocellular (liver)cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma,islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, laryngealcancer, leukemia acute lymphoblastic cancer, leukemia acute myeloidcancer, leukemia chronic lymphocytic cancer, leukemia chronicmyelogenous cancer, leukemia hairy cell cancer, liver cancer, lungcancer non-small cell, lung cancer small cell, male breast cancer,malignant mesothelioma, medulloblastoma, melanoma, merkel cellcarcinoma, multiple endocrine neoplasia syndrome, mycosis fungoides,myeloma multiple, nasal cavity, paranasal and sinus cancer,nasopharyngeal cancer, neuroblastoma, oral cavity and lip cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian epithelial cancer, ovarian germ cell tumor, pancreaticcancer, parathyroid cancer, penile cancer, pheochromocytoma, pineal andsupratentorial primitive neuroectodermal tumors, pituitary tumor,pleuropulmonary blastoma, prostate cancer, rectal cancer, renal, pelvisand ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, sarcoma soft tissue adult, Sezary syndrome, skincancer, small intestine cancer, stomach (gastric) cancer, testicularcancer, thymoma, thyroid cancer, urethral cancer, uterine cancerendometrial, vaginal cancer, vulvar cancer, Waldenstrom'smacroglobulinemia, and Wilms' tumor., other than an inflammatorydisease; other than an inflammatory disease that is associated withmicroglial cell activation or stimulation., a proliferative disease., aproliferative disease is malignant, pre-malignant or benign cancer, adisease resulting from or is associated an infection selected from thegroup consisting of HIV infection, HPV infection and HSV infection. 2.The method of claim 1, wherein the composition comprises a carrier orexcipient selected from the group consisting of dimethyl sulfoxide(DMSO), phosphate buffered saline, saline, a lipid based formulation, aliposomal formulation, a nanoparticle formulation, a micellarformulation, a water soluble formulation, and a biodegradable polymer.3. The method of claim 1, wherein R₁ and R₂ are independently —CH₃,—(C═O)CH₂N(CH₃)₂ or —(C═O)CH₂N⁺H(CH₃)₂.Cl⁻ and R₈ and R₉ areindependently —OCH₃, —O(C═O)CH₂N(CH₃)₂ or —O(C═O)CH₂N⁺H(CH₃)₂.Cl⁻ orwherein R₁ and R₂ are independently —CH₃ and R₈ and R₉ are independently—OCH₃.
 4. The method of claim 1, wherein the pharmaceutically acceptablecarrier or excipient comprises dimethyl sulfoxide or at least onedietary fat or oil selected from the group consisting of castor oil andpeanut oil.
 5. The method of claim 1, wherein the composition isadministered to a human in an amount of about 10 mg/kg to about 375mg/kg per dose.
 6. The method of claim 1, wherein the composition isadministered on a schedule selected from the group consisting of: daily,daily for 5 or more days to a week, daily for 5 or more days to 2 weeks,daily for 5 or more days to 3 weeks.
 7. A method of treating leukemia ina subject comprising: a) administering a composition consistingessentially of one catecholic and a pharmaceutically acceptable carrieror excipient, wherein the wherein the catecholic butane has the formula:

wherein R₁ and R₂ are independently —H, a lower alkyl, a lower acyl, analkylene or an unsubstituted or substituted amino acid residue or saltthereof; R₃, R₄, R₅, R₆, R₁₀, R₁₁, R₁₂ and R₁₃ are independently —H or alower alkyl; and R₇, R₈ and R₉ are independently —H, —OH, a loweralkoxy, a lower acyloxy, or any two adjacent groups together may be analkyene dioxy, or an unsubstituted or substituted amino acid residue orsalt thereof, provided that the catecholic butane is not NDGA; and (b)administering the composition to the subject systemically by a route ofadministration selected from the group consisting of: oraladministration; inhalation administration; intra-arterialadministration, with or without occlusion; intracranial administration;intraventricular administration; intravenous administration;intramuscular administration; implantation administration; and centralvenous administration.
 8. The method of claim 7, wherein R₁ and R₂ areindependently —CH₃, —(C═O)CH₂N(CH₃)₂ or —(C═O)CH₂N⁺H(CH₃)₂.Cl⁻ and R₈and R₉ are independently —OCH₃, —O(C═O)CH₂N(CH₃)₂ or—O(C═O)CH₂N⁺H(CH₃)₂.Cl⁻ or wherein R₁ and R₂ are independently —CH₃ andR₈ and R₉ are independently —OCH₃.
 9. The method of claim 7 wherein theleukemia is selected from acute lymphoblastic leukemia, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia;hairy cell leukemia; leukemia acute myeloid cancer, leukemia chroniclymphocytic cancer, leukemia chronic myelogenous cancer, leukemia hairycell cancer.
 10. The method of claim 7, wherein the pharmaceuticallyacceptable carrier or excipient is an oil.
 11. The method of claim 7,wherein the composition is administered on a schedule selected from thegroup consisting of: daily, daily for 5 or more days to a week, dailyfor 5 or more days to 2 weeks, daily for 5 or more days to 3 weeks. 12.The method of claim 7, wherein the amount of chatecholic butaneadministered is at least 30 mg per dose.
 13. The method of claim 7wherein the amount of chatecholic butane administered is at least 90 mgper dose.
 14. The method of claim 7, wherein the chatecholic butane ispresent in the composition at a concentration of 20 mg/mL.
 15. Themethod of claim 7, wherein the pharmaceutically acceptable carrier orexcipient comprises Cremaphor EL, ethanol and saline.
 16. The method ofclaim 8, wherein the Cremaphor EL concentration is 6%.
 17. The method ofclaim 8, wherein the ethanol concentration is 6%.
 18. The method ofclaim 8, wherein the composition administered to the subject comprisesat least 2 mg of per dose.
 19. The method of claim 8, wherein thecomposition is administered to a human in an amount of about 10 mg/kg toabout 375 mg/kg per dose.
 20. A method of treating leukemia in a subjectcomprising: a) administering a composition consisting essentially oftetra-O-methyl NDGA and a pharmaceutically acceptable carrier orexcipient, and (b) administering the composition to the subjectsystemically by a route of administration selected from the groupconsisting of: oral administration; inhalation administration;intra-arterial administration, with or without occlusion; intracranialadministration; intraventricular administration; intravenousadministration; intramuscular administration; implantationadministration; and central venous administration.